Compositions and methods for regulating erythropoeitin expression and ameliorating anemia and stimulating erythropoiesis

ABSTRACT

The invention provides compositions (e.g., pharmaceuticals, formulations) and methods for ameliorating (e.g., preventing or treating) an anemia and/or stimulating erythropoiesis and/or EPO erythropoietin synthesis. The invention provides compositions comprising a chimeric protein artificial transcription factor comprising a plurality of (multiple) protein DNA-binding domains, e.g., zinc finger binding domains, specific for the promoter region of an erythropoietin (EPO) gene; a consensus nuclear localization protein sequence; a cell-penetrating peptide sequence; and a transcription activation domain.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/019,815, filed Jan. 8, 2008. Theaforementioned application is expressly incorporated herein by referencein its entirety and for all purposes.

TECHNICAL FIELD

This invention relates to molecular and cellular biology, biochemistry,molecular genetics, protein therapy, and drug design and discovery. Theinvention provides compositions (e.g., pharmaceuticals, formulations)and methods for ameliorating (e.g., preventing or treating) anemiaand/or stimulating erythropoiesis. The invention provides compositionscomprising a chimeric protein artificial transcription factor comprisinga plurality of (multiple) protein DNA-binding domains, e.g., zinc fingerbinding domains, specific for the promoter region of an erythropoietin(EPO) gene; a consensus nuclear localization protein sequence; acell-penetrating peptide sequence; and, (4) a transcription activationdomain.

BACKGROUND

Erythropoietin (EPO, or hematopoietin or hemopoietin) is a glycoproteinhormone that is a cytokine for erythrocyte (red blood cell) precursorsin the bone marrow. EPO is produced by the kidney. EPO is the hormonethat regulates the red blood cell production (erythropoiesis), and lackof EPO can cause anemia.

SUMMARY

The invention provides compositions and methods for ameliorating anemiaand/or stimulating erythropoiesis and EPO synthesis. The inventionprovides chimeric proteins, or compositions comprising chimericproteins, that act as artificial transcription factors, comprising:

-   -   (a) (1) a plurality of (multiple) EPO-specific DNA-binding        domains (e.g., zinc finger DNA-binding domains) specific for the        promoter region of (or any transcriptional regulatory region of)        an erythropoietin (EPO) gene or a Erythropoietin Stimulating        Protein (NESP) gene; (2) a consensus nuclear localization        protein sequences; 3) a cell-penetrating peptide sequence;        and, (4) a transcription activation domain;    -   (b) the chimeric protein of (a), wherein the chimeric protein        comprises two, three, four, five, six or more EPO-specific        DNA-binding domains (e.g., zinc finger DNA-binding domains)        specific for an EPO gene;    -   (c) the chimeric protein of (a) or (b), wherein the EPO gene        comprises a mammalian, e.g., a mouse and/or a human, EPO gene;    -   (d) the chimeric protein of any of (a) to (c), wherein the        chimeric protein comprises one, two, three, four, five, six or        more consensus nuclear localization protein sequences;    -   (e) the chimeric protein of any of (a) to (c), wherein the        chimeric protein comprises one, two, three, four, five, six or        more transcription activation domains; or    -   (f) the chimeric protein of any of (a) to (e), wherein the        chimeric protein comprises a formulation for subcutaneous,        parenteral, topical, oral or local administration, or for        aerosol or transdermal administration.

The invention provides recombinant or synthetic nucleic acids comprisingnucleic acid sequence encoding a chimeric protein of this invention.

The invention provides vectors, plasmids, recombinant viruses orexpression vehicles comprising a nucleic acid of this invention.

The invention provides pharmaceutical formulations comprising

-   -   (a) a chimeric protein of this invention; a nucleic acid of this        invention; and/or the vector, plasmid, recombinant virus or        expression vehicle of this invention; or,    -   (b) the pharmaceutical formulation of (a) formulated for        subcutaneous, parenteral, topical, oral or local administration,        or for aerosol or transdermal administration.

The invention provides methods for ameliorating an anemia in anindividual comprising:

-   -   (a) (i) a pharmaceutical formulation of this invention; a        chimeric protein of this invention; a nucleic acid of this        invention; and/or a vector, plasmid, recombinant virus or        expression vehicle of this invention; and    -   (ii) administering an effective amount of the composition of (a)        to an individual in need thereof;    -   (b) the method of (a), wherein the individual is a mammal (e.g.,        a mouse, a human);    -   (c) the method of (a) or (b), wherein the composition comprises        a pharmaceutical formulation;    -   (d) the method of (c), wherein the pharmaceutical formulation        comprises a formulation for subcutaneous, parenteral, topical,        oral or local administration, or for aerosol or transdermal        administration; or    -   (e) the method of any of (a) to (d), wherein the anemia is        caused by cancer, or a cancer therapeutic or therapy.

The invention provides kits comprising (a) (i) the chimeric protein ofthis invention; the nucleic acid of this invention; and/or the vector,plasmid, recombinant virus or expression vehicle of this invention; and,(b) instructions to perform the method of this invention. Also providedare kits comprising instructions for practicing the methods of theinvention.

The invention provides chimeric proteins comprising:

-   -   (a) (1) a plurality of (multiple) DNA-binding domains specific        for (that can specifically bind to) a promoter and/or another        transcriptional regulatory region of an erythropoietin (EPO)        gene or a Erythropoietin Stimulating Protein (NESP) gene;    -   (2) at least one nuclear localization peptide (NLP) domain;    -   (3) at least one cell-penetrating peptide (CPP); and,    -   (4) at least one transcription activation (TA) domain; or    -   (b) the chimeric protein of (a), comprising an EPO-specific zinc        finger DNA-binding domain, or an NESP-specific zinc finger        DNA-binding domain.

In alternative embodiments, the chimeric protein, the EPO- orNESP-specific DNA-binding domain, the NLP domain, the CPP domain and/orthe TA domain comprises or consist of a recombinant protein, a syntheticprotein, a peptidomimetic, a non-natural peptide or a combinationthereof.

In alternative embodiments, the EPO or NESP gene or transcriptionalregulatory region of an EPO or NESP gene comprises (or consists of): (a)a mammalian EPO or NESP gene or transcriptional regulatory region or (b)a mouse or a human EPO or NESP gene or transcriptional regulatoryregion. The transcriptional regulatory region can comprise (or consistof) a promoter or an enhancer, an EPO or NESP promoter or EPO or NESPenhancer, or a synthetic promoter. The chimeric protein can comprise (orconsist of) multiple copies of the EPO- or NESP-specific DNA-bindingdomain, the NLP, the CPP and/or the TA domain. In alternativeembodiments, the chimeric protein comprises (or consists of) two, three,four, five, six or more EPO- or NESP-specific DNA-binding domainsspecific for (that can specifically bind to) a promoter and/or anothertranscriptional regulatory region of an EPO or NESP gene. The chimericprotein can comprises (or consists of) one, two, three, four, five, sixor more nuclear localization peptide (NLP) domains or consensus nuclearlocalization proteins.

In alternative embodiments: the chimeric protein comprises one, two,three, four, five, six or more cell-penetrating peptides (CPPs); or, thechimeric protein comprises one, two, three, four, five, six or more TAdomains, and/or one or more other functional domains with a histoneacetyltransferase (HAT) activity. The at least one TA domain cancomprise a herpes simplex virus (HSV) VP-16 activation peptide domain ora peptide derived from the C-terminal transcription activation domain ofβ-catenin (FDTDL). The at least one (the one or more) zinc fingerDNA-binding domain(s) can comprise: (1) a zinc-finger of the C₂H₂ class;(2) a zinc-finger of the C₄ class; or (3) a zinc-finger of C₆ class. Theat least one (the one or more) zinc finger DNA-binding domain(s) cancomprise (or consist of) the consensus sequenceCys-X₂₋₄-Cys-X₃-Phe-X₅-Leu-X₂-His-X3-His.

In alternative embodiments: the at least one nuclear localizationpeptide (NLP) domain comprises: (1) an NLP sequence of a large T antigenof the simian virus 40 (SV-40), or PKKKRKV (SEQ ID NO:2); (2) aconsensus sequence fitting B₄, P(B₃X), PXX(B₃X), B₃(H/P), where B is abasic amino acid, P is proline, H is histidine, X is any amino acid andletters in parentheses can be in any order; (3) a bipartite NLPcomprising two short stretches of basic amino acids separated by anon-conserved sequence; or (4) a cellular nucleoplasmin proteinKRPAATKKAGQAKKKK (SEQ ID NO:4).

In alternative embodiments: the at least one cell-penetrating peptide(CPP) comprises: (1) a plurality of polycationic amino acid residues;(2) a plurality of arginine amino acid residues; or (3) a TAT protein(Trans-acting Activator of Transcription) of a Human ImmunodeficiencyVirus (HIV-1).

In alternative embodiments: (1) the at least one TA domain is at leastapproximately 25% hydrophobic and is linked to the at least one zincfinger DNA-binding domain in a manner that does not interfere with thepromoter or a transcriptional regulatory binding activity of the zincfinger DNA binding peptide, and the TA domain is both necessary andsufficient to activate transcription of the gene; and/or (2) the TAdomain is between about 5 to 25 amino acids in length, or is betweenabout 6 to 20 amino acids in length, or is about 5, 6, 7, 8, 9, 10, 11,11, 12, 13, 14 or 15 amino acids in length.

In alternative embodiments: the at least one TA domain comprises aherpes simplex virus (HSV) VP-16 activation peptide domain or a peptidederived from the C-terminal transcription activation domain of β-catenin(FDTDL).

In alternative embodiments: at least one, or all, of the domains and/orchimeric proteins further comprises, or is attached to, a lipid, ahydrophobic alkane or alkene (olefin) moiety, or a polyethylene glycol(PEG) moiety. The at least one, several, or all, of the chimericproteins can further comprises, or be attached to, an epitope peptidetag or a detectable composition or moiety. The detectable composition ormoiety can comprise a phosphoprotein, a fluorescent molecule, afluorescent tagged protein, a radiolabel or a radiolabeled protein.

In alternative embodiments chimeric proteins of the invention canfurther comprise a small molecule, a hormone or a cytokine thatincreases or upregulates erythropoiesis or red blood cell production ina mammalian.

In alternative embodiments chimeric proteins of the invention cancomprise (or be formulated or made as): (a) a formulation forsubcutaneous, parenteral, topical, oral or local administration, or foraerosol or transdermal administration, or administration by nebulizer;or (b) the chimeric protein of (a), wherein the topical formulationcomprises an ointment, a cream, a powder, an emulsion, a gel, aglycerogelatin, a paste, a plaster, a sprayable composition or a lotion.

The invention provides compositions comprising a plurality of one ormore chimeric proteins of the invention, or a formulation of theinvention. The composition can further comprise a small molecule, ahormone or a cytokine that increases or upregulates erythropoiesis orred blood cell production in a mammalian. The composition can furthercomprise a synthetic or a recombinant erythropoietin, e.g., a humansynthetic or a recombinant erythropoietin.

The invention provides liquids, gels, hydrogels, powders or aqueousformulations comprising one or more chimeric proteins of the invention,or a composition or formulation of the invention.

The invention provides vesicles, liposomes, nanoparticles or nanolipidparticles (NLP) comprising one or more chimeric proteins of theinvention, or a composition or formulation of the invention, or aliquid, gel, hydrogel, powder or aqueous formulation of the invention.

The invention provides cells, e.g., isolated, host or cultured cells,comprising (or having contained therein) one or more chimeric proteinsof the invention (or a nucleic acid, such as a vector, encoding achimeric protein of the invention), or a composition or formulation ofthe invention, or a liquid, gel, hydrogel, powder or aqueous formulationof the invention. These cells, e.g., isolated, host or cultured cells,can be mammalian cells, such as human cells, a non-human primate cell, amonkey cell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell,a hamster cell, a goat cell, a bovine cell, an equine cell, an ovinecell, a canine cell or a feline cell. These cells, e.g., isolated, hostor cultured cells, also can be prokaryotic cells, yeast cells, fungalcells, insect cells or plant cells.

The invention provides formulations, e.g., pharmaceutical or sterileformulations, comprising one or more chimeric proteins of the invention(or a nucleic acid, such as a vector, encoding a chimeric protein of theinvention), or a composition or formulation of the invention, or aliquid, gel, hydrogel, powder or aqueous formulation of the invention, avesicle, liposome, nanoparticle or nanolipid particle (NLP) of theinvention, or an isolated or cultured cell of the invention.

The invention provides products of manufacture comprising one or morechimeric proteins of the invention (or a nucleic acid, such as a vector,encoding a chimeric protein of the invention), or a composition orformulation of the invention, or a liquid, gel, hydrogel, powder oraqueous formulation of the invention, a vesicle, liposome, nanoparticleor nanolipid particle (NLP) of the invention, or an isolated or culturedcell of the invention.

The invention provides recombinant or synthetic nucleic acids (e.g.,including vectors, plasmids, recombinant viruses or expression vehicles)comprising a nucleic acid sequence encoding a chimeric protein of theinvention. The invention provides vectors, plasmids, recombinant virusesand expression vehicles comprising: (a) a nucleic acid of the invention;(b) the vector, plasmid, recombinant virus or expression vehicle of (a),wherein the nucleic acid is operatively linked to a constitutivepromoter or an inducible promoter; or (c) the vector, plasmid,recombinant virus or expression vehicle of (a) or (b), wherein thepromoter is only active in a hematopoietic cell.

The invention provides cells, e.g., isolated, host or cultured cells,comprising (or having contained therein) a nucleic acid of theinvention, or a vector, plasmid, recombinant virus or expression vehicleof the invention. These cells, e.g., isolated or cultured cells, can bemammalian cells, such as human cells, a non-human primate cell, a monkeycell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, ahamster cell, a goat cell, a bovine cell, an equine cell, an ovine cell,a canine cell or a feline cell. These cells, e.g., isolated or culturedcells, also can be prokaryotic cells, yeast cells, fungal cells, insectcells or plant cells.

The invention provides formulations, including pharmaceutical or sterileformulations, comprising:

-   -   (a) (i) a chimeric protein of the invention, a liquid, gel,        hydrogel, powder or aqueous formulation of the invention, a        vesicle, liposome, nanoparticle or nanolipid particle (NLP) of        the invention, or an isolated, host or cultured cell of the        invention, and (ii) a pharmaceutically acceptable excipient;    -   (b) the pharmaceutical formulation or sterile formulation of (a)        formulated for subcutaneous, parenteral, topical, oral or local        administration, or for aerosol or transdermal administration, or        administration by a nebulizer; or    -   (c) the pharmaceutical formulation or sterile formulation of (a)        is formulated for topical application in the form of an        ointment, a cream, a powder, an emulsion, a gel, a        glycerogelatin, a paste, a plaster, a sprayable composition or a        lotion.

The invention provides methods for ameliorating or preventing an anemia,and/or stimulating erythropoiesis and/or erythropoietin (EPO) synthesis,in an individual comprising: (a) providing: a pharmaceutical or sterileformulation of the invention; a pharmaceutical formulation of theinvention; a chimeric protein of the invention; a liquid, gel, hydrogel,powder or aqueous formulation of the invention; a vesicle, liposome,nanoparticle or nanolipid particle (NLP) of the invention; an isolated,host or cultured cell of the invention; a nucleic acid of the invention;or a vector, plasmid, recombinant virus or expression vehicle of theinvention; and (b) administering an effective amount of (a) to anindividual in need thereof. The individual in need thereof can be amammalian or a human. The anemia ameliorated or prevented can be causedby a genetic disorder, an infection, a dietary disorder or deficiency, apollutant, a pesticide, herbicide or insecticide, a poison, a venom, atoxin, a biological agent, a drug, a cancer or a cancer therapeutic orcancer therapy. In one aspect of the method, the anemia ameliorated orprevented is a microcytic, normocytic or macrocytic form of anemia. Theanemia ameliorated or prevented can be: a drug-induced anemia; caused byan infection; caused by an iron deficiency; caused by rhesus disease(hemolytic disease of newborn); caused by sickle-cell disease,thalassemia or Plummer-Vinson syndrome (PVS, also calledPaterson-Brown-Kelly syndrome or sideropenic dysphagia); a sideroblasticanemia-congenital or acquired; caused by Gaucher's disease; caused by avitamin deficiency; caused by autoimmune hemolytic anemia (AIHA); causedby a cancer; or, caused by heavy metal poisoning or pyridoxinedeficiency. The vitamin deficiency can be a folate or B12 deficiency(pernicious anemia or Addison's anemia). The drug-induced anemia can becaused by methyldopa or fludarabin. The AIHA can be caused by Systemiclupus erythematosus, a drug, Evans syndrome, chronic lymphocyticleukemia or is idiopathic. The cancer can be chronic lymphocyticleukemia, small cell lymphoma (or small lymphocytic lymphoma) or anon-Hodgkin's lymphoma; or the anemia is caused by myelophythisissecondary to an acute megakaryoblastic leukemia, a lymphoma, a myelomaor a carcinoma metastatic to bone marrow. In alternative embodiments ofmethods of the invention, the infection is an EBV infectiousmononucleosis, a Babesiosis infection, or equine infectious anemia. Thecancer therapeutic or cancer therapy can be radiotherapy, hormonetherapy or chemotherapy. The heavy metal poisoning can be leadpoisoning, mercury poisoning (hydrargaria), copper poisoning, nickelpoisoning, manganese poisoning (manganism) or cadmium poisoning.

The invention provides kits comprising a pharmaceutical or sterileformulation of the invention; a pharmaceutical formulation of theinvention; a chimeric protein of the invention; a liquid, gel, hydrogel,powder or aqueous formulation of the invention; a vesicle, liposome,nanoparticle or nanolipid particle (NLP) of the invention; an isolated,host or cultured cell of the invention; a nucleic acid of the invention;or a vector, plasmid, recombinant virus or expression vehicle of theinvention.

The invention provides uses of a pharmaceutical or sterile formulationof the invention; a chimeric protein of the invention; a liquid, gel,hydrogel, powder or aqueous formulation of the invention; a vesicle,liposome, nanoparticle or nanolipid particle (NLP) of the invention; anisolated, host or cultured cell of the invention; a nucleic acid of theinvention; or a vector, plasmid, recombinant virus or expression vehicleof the invention, to make a pharmaceutical composition for amelioratingor preventing an anemia in an individual. In alternative embodiments,the individual in need thereof is a mammal or a human. The anemiaameliorated or prevented can be caused by a genetic disorder, aninfection, a dietary disorder or deficiency, a pollutant, a pesticide,herbicide or insecticide, a poison, a venom, a toxin, a biologicalagent, a drug, a cancer or a cancer therapeutic or cancer therapy. Theanemia ameliorated or prevented can be a microcytic, normocytic ormacrocytic form of anemia. The anemia ameliorated or prevented can be: adrug-induced anemia; caused by an infection; caused by an irondeficiency; caused by rhesus disease (hemolytic disease of newborn);caused by sickle-cell disease, thalassemia or Plummer-Vinson syndrome(PVS, also called Paterson-Brown-Kelly syndrome or sideropenicdysphagia); a sideroblastic anemia-congenital or acquired; caused byGaucher's disease; caused by a vitamin deficiency; caused by autoimmunehemolytic anemia (AIHA); caused by a cancer; or, caused by heavy metalpoisoning or pyridoxine deficiency. The vitamin deficiency can be afolate or B12 deficiency (pernicious anemia or Addison's anemia). Thedrug-induced anemia can be caused by methyldopa or fludarabin. The AIHAcan be caused by Systemic lupus erythematosus, a drug, Evans syndrome,chronic lymphocytic leukemia or is idiopathic. The cancer can be chroniclymphocytic leukemia, small cell lymphoma (or small lymphocyticlymphoma) or a non-Hodgkin's lymphoma; or the anemia is caused bymyelophythisis secondary to an acute megakaryoblastic leukemia, alymphoma, a myeloma or a carcinoma metastatic to bone marrow. Theinfection can be an EBV infectious mononucleosis, a Babesiosisinfection, or equine infectious anemia. The cancer therapeutic or cancertherapy can be radiotherapy, hormone therapy or chemotherapy. The heavymetal poisoning can be lead poisoning, mercury poisoning (hydrargaria),copper poisoning, nickel poisoning, manganese poisoning (manganism) orcadmium poisoning.

The invention provides methods for upregulating or activating anerythropoietin (EPO) gene in a cell comprising: (a) providing: apharmaceutical or sterile formulation of the invention; a chimericprotein of the invention; a liquid, gel, hydrogel, powder or aqueousformulation of the invention; a vesicle, liposome, nanoparticle ornanolipid particle (NLP) of the invention; a isolated, host or culturedcell of the invention; a nucleic acid of the invention; a product ofmanufacture of the invention; or a vector, plasmid, recombinant virus orexpression vehicle of the invention; and (b) contacting or administeringan effective amount of (a) to the cell, thereby upregulating oractivating an erythropoiesis gene in a cell. The cell can be a mammaliancell, e.g. such as a human cell, a non-human primate cell, a monkeycell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, ahamster cell, a goat cell, a bovine cell, an equine cell, an ovine cell,a canine cell or a feline cell. In alternative embodiments, thecontacting or administering is in vitro, ex vivo or in vivo.

The invention provides methods for making a recombinant erythropoietin(EPO) in a cell comprising: (a) providing: a pharmaceutical or sterileformulation of the invention; a chimeric protein of the invention; aliquid, gel, hydrogel, powder or aqueous formulation of the invention; avesicle, liposome, nanoparticle or nanolipid particle (NLP) of theinvention; a isolated, host or cultured cell of the invention; a nucleicacid of the invention; a product of manufacture of the invention; or avector, plasmid, recombinant virus or expression vehicle of theinvention; (b) inserting in a cell a nucleic acid comprising aheterologous EPO coding sequence operatively linked to an EPO specificpromoter; and (c) contacting or administering an effective amount of (a)to the cell, thereby making a recombinant EPO in the cell. The cell canbe a mammalian cell, e.g., such as a human cell, a non-human primatecell, a monkey cell, a mouse cell, a rat cell, a guinea pig cell, arabbit cell, a hamster cell, a goat cell, a bovine cell, an equine cell,an ovine cell, a canine cell or a feline cell. The contacting oradministering can be in vitro, ex vivo or in vivo.

The invention provides methods for increasing hematocrit in a mammalcomprising: (a) providing: a pharmaceutical or sterile formulation ofthe invention; a chimeric protein of the invention; a liquid, gel,hydrogel, powder or aqueous formulation of the invention; a vesicle,liposome, nanoparticle or nanolipid particle (NLP) of the invention; aisolated, host or cultured cell of the invention; a nucleic acid of theinvention; a product of manufacture of the invention; or a vector,plasmid, recombinant virus or expression vehicle of the invention; and(b) contacting or administering an effective amount of (a) to themammal, thereby increasing the mammal's hematocrit. The cell can be amammalian cell, e.g., such as a human cell, a non-human primate cell, amonkey cell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell,a hamster cell, a goat cell, a bovine cell, an equine cell, an ovinecell, a canine cell or a feline cell. The contacting or administeringcan be in vitro, ex vivo or in vivo.

The invention provides methods for increasing endogenous erythropoietin(EPO) production in an individual or cell in need thereof, the methodcomprising: (a) providing: a pharmaceutical or sterile formulation ofthe invention; a chimeric protein of the invention; a liquid, gel,hydrogel, powder or aqueous formulation of the invention; a vesicle,liposome, nanoparticle or nanolipid particle (NLP) of the invention; aisolated, host or cultured cell of the invention; a nucleic acid of theinvention; a product of manufacture of the invention; or a vector,plasmid, recombinant virus or expression vehicle of the invention; and(b) contacting or administering an effective amount of (a) to theindividual, thereby increasing endogenous erythropoietin (EPO)production. The cell can be a mammalian cell, e.g., such as a humancell, a non-human primate cell, a monkey cell, a mouse cell, a rat cell,a guinea pig cell, a rabbit cell, a hamster cell, a goat cell, a bovinecell, an equine cell, an ovine cell, a canine cell or a feline cell. Thecontacting or administering can be in vitro, ex vivo or in vivo.

In alternative embodiments, the individual can be a human or an animal.The effective amount of (a) can be administered to the individual totreat, ameliorate and/or prevent: myelodysplastic syndrome; a peripheralnerve injury; a hemoglobin H disease; a testicular torsion-detorsion;post-infarct myocardial damage; or a neurologic disorder; and/or, totreat or ameliorate a wound and/or an inflammation, or to treat orameliorate conjunctivitis; surgical or accidental wounds; a bedsore; aburn; an inflammation of the skin, mucous membranes, airways or lungs;an eczema or a skin disorder accompanied by necrosis, by dermatitis, bypsoriasis or by diabetes mellitus. The effective amount of (a) can beadministered as a neuroprotective in low birth weight infants. Theeffective amount of (a) can be administered as a neuroprotective foraltitude related illnesses. The neurologic disorder can be ischemicstroke, intracerebral hemorrhage, subarachnoid hemorrhage, traumaticbrain injury or Parkinson's disease.

The invention provides use of a pharmaceutical or sterile formulation ofthe invention; a chimeric protein of the invention; a liquid, gel,hydrogel, powder or aqueous formulation of the invention; a vesicle,liposome, nanoparticle or nanolipid particle (NLP) of the invention; aisolated, host or cultured cell of the invention; a nucleic acid of theinvention; a product of manufacture of the invention; or a vector,plasmid, recombinant virus or expression vehicle of the invention, tomake a pharmaceutical composition to: (a) treat, ameliorate and/orprevent: a myelodysplastic syndrome; a peripheral nerve injury; ahemoglobin H disease; a testicular torsion-detorsion; post-infarctmyocardial damage; or a neurologic disorder; (b) act as aneuroprotective in low birth weight infants; (c) as a neuroprotectivefor altitude related illnesses; or (d) to treat or ameliorate a woundand/or an inflammation, or to treat or ameliorate conjunctivitis;surgical or accidental wounds; a bedsore; a burn; an inflammation of theskin, mucous membranes, airways or lungs; an eczema or a skin disorderaccompanied by necrosis, by dermatitis, by psoriasis or by diabetesmellitus. The neurologic disorder can be ischemic stroke, intracerebralhemorrhage, subarachnoid hemorrhage, traumatic brain injury orParkinson's disease.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. All publications,patents, patent applications cited herein are hereby expresslyincorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 illustrates photomicrograph images that confirms that chimericproteins (also called “Designed Regulatory Proteins”, or DRPs) of thisinvention can enter mammalian cells, using in this example humanlymphocyte cells: left two panels illustrate a fluorescence image, andright two panels illustrate a phase contrast image; the upper two panelsillustrate detection of green fluorescent protein expressed by chimericproteins (DRPs) of the invention, as discussed in detail in Example 1,below.

FIG. 2 graphically illustrates data from an ELISA assay demonstratingthat chimeric proteins (DRPs) of this invention (the exemplary EPO1-DRPand EPO2-DRP) when inserted into mammalian cells can enhance secretionof EPO protein in those cells, as discussed in detail in Example 1,below.

FIG. 3 graphically illustrates data demonstrating blood EPO levels inmice after administration of exemplary chimeric proteins (DRPs) of thisinvention (EPO1-DRP and EPO2-DRP); as discussed in detail in Example 1,below.

FIG. 4 schematically illustrates exemplary target DNA sequences forexemplary chimeric proteins (DRPs) of this invention, particularly, thezinc finger DNA-binding domains of chimeric proteins (DRPs) of thisinvention; as discussed in detail in Example 2, below.

FIG. 5 illustrates alternative exemplary zinc finger DNA targetsequences for chimeric proteins (DRPs) of the invention, with thecorresponding exemplary zinc finger DNA-binding motif; as discussed indetail in Example 2, below.

FIG. 6 illustrates exemplary chimeric proteins comprising one or more orall of the listed amino acid zinc finger DNA-binding motifs; asdiscussed in detail in Example 2, below.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION

The invention provides compositions and methods for the prevention oramelioration, including the treatment of, anemia and/or stimulatingerythropoiesis and EPO synthesis. The invention provides compositionsand methods for the amelioration or prevention of anemia caused by anydisease or condition, including anemic phenotypes caused by anygenotype, e.g., any genetic predisposition, such as a mutation in a genenecessary to maintain normal erythropoiesis and/or red blood cellcounts. However, in alternative embodiments the anemias ameliorated orprevented are not necessarily caused by a particular genotype, butrather can be caused by exposure to an environmental agent, e.g., adrug, food, insecticide, herbicide, pesticide, poison, venoms, toxin,biological warfare agent, pollutant, allergen and the like. In someembodiments, a combination of these elements cause the anemia, e.g., oneor more environmental agents and a genetic predisposition. Inalternative embodiments the anemias ameliorated or prevented are causedby a disease or condition, e.g., cancer, folate or B12 deficiency,autoimmune hemolytic anemia and the like.

The invention provides compositions and methods for a proteintherapeutic-based approach for manipulating, e.g., stimulating orinhibiting, the expressing of erythropoietin (EPO) coding sequences,including EPO genes. In one embodiment, compositions and methods of theinvention are used to stimulate an endogenous and/or exogenous orheterologous mammalian, e.g., human, erythropoietin (EPO) gene, wherethe EPO gene is stimulated by compositions comprising chimeric proteins(DRPs) of the invention. For example, in one embodiment, a chimericprotein (DRP) of the invention is used ex vivo or in vivo to stimulatean exogenous or heterologous mammalian EPO gene inserted into a cell,including e.g. a transgenic animal comprising an exogenous orheterologous EPO gene, or cells with exogenous or heterologous mammalianEPO genes implanted in an animal or individual.

In alternative embodiments, chimeric proteins (DRPs) of this inventioncomprise (or consist of) an artificial transcription factor comprising(or consisting of) (1) one or multiple (two, three, four, five, six ormore) EPO-specific DNA-binding domains (e.g., zinc finger DNA-bindingdomains) that can bind to an EPO gene transcriptional regulatorysequence and/or an Erythropoietin Stimulating Protein (NESP) genetranscriptional regulatory sequence; such as a promoter and/or anenhancer, and in one embodiment, the zinc finger DNA-binding domain isspecific for an EPO gene and/or an Erythropoietin Stimulating

Protein (NESP) gene; 2) one or multiple consensus nuclear localizationsequences; 3) one or multiple cell-penetrating peptide sequences and 4)one or multiple transcription activation (TA) domains.

Alternative embodiments include nucleic acids that encode the chimericproteins (DRPs) of this invention, including expression cassettes,plasmids, vectors and the like comprising these DPR-coding sequences.Alternative embodiments include host cells, e.g., cultured, isolated ortransformed cells, comprising nucleic acids and/or chimeric proteins ofthis invention.

In alternative embodiments, the erythropoietin (EPO) coding sequence(e.g., a gene) activated by a chimeric protein (DRP) of this inventionis endogenous and/or exogenous or heterologous; for example, a cell canbe supplemented with an exogenous or heterologous EPO coding sequence(e.g., a gene) and administration of a chimeric protein of the inventionor a nucleic acid encoding a chimeric protein of the invention activatesthe exogenous or heterologous EPO coding sequence, the endogenous EPOcoding sequence or both.

In one aspect of the invention, compositions comprise EPO gene-specificchimeric proteins (e.g., the chimeric proteins of the invention cantarget transcriptional activators such as promoter of EPO genes). In oneembodiment, these compositions of the invention are used as a therapyfor humans and other mammals with anemia (e.g., to treat or ameliorateanemia), or to prevent anemia. In one embodiment, after therapy with anEPO-specific DRP-comprising composition of this invention (e.g., apharmaceutical composition), EPO gene transcription occurs (EPO codingsequence is generated). In one embodiment, EPO protein is secreted froman individual's, e.g., a patient, a human, any mammal. In oneembodiment, EPO protein is secreted from cells and enters theindividual's bloodstream in clinically significant amounts. In oneembodiment, EPO protein is secreted from the individual's intestinalcells and enters the bloodstream in clinically significant amounts. Inone embodiment, chimeric proteins of this invention, including e.g.,compositions comprising these DRPs, are used for mammalian, e.g., human,erythropoietin (EPO) manipulation, e.g., activation or upregulation, andcan include pharmaceutical preparations (formulations) to activate theendogenous and/or an exogenous EPO gene in vitro, ex vivo and/or invivo.

In one embodiment, the invention comprises selecting a target site inthe EPO gene, e.g., an EPO promoter or other transcriptional regulatorysequence, that can be specifically bound by a DNA-binding domain, e.g.,a zinc finger DNA-binding domain, and then designing and manufacturing achimeric (e.g., a recombinant) protein that will bind that EPO DNAsequence with high affinity and specificity.

In one embodiment, the invention provides a chimeric (e.g., arecombinant) protein with the ability to enter cells (e.g., human,mammalian or other cells), go to the nucleus, bind specifically to theEPO gene (or equivalent), and activate transcription of the EPO gene (orequivalent), resulting in accumulation of EPO (or equivalent) in thecell and/or secretion of EPO (or equivalent) from the cell, andalternatively, secretion or release of EPO (or equivalent) into thebloodstream.

In one embodiment, chimeric proteins of this invention comprise aplurality (e.g., two, three, four or more) domains that enable each ofat least four activities: at least one (one or more) proteintransduction domain(s); at least one (one or more) nuclear localizationsignal domain(s); at least one (one or more) EPO specific DNA-bindingdomain(s) (e.g., comprising one, two, three, four, five, six or morezinc fingers), and at least one (one or more) transactivation domain(s).

Polypeptides and Peptides

The invention provides chimeric polypeptides for ameliorating (e.g.,preventing or treating) anemia and/or stimulating erythropoiesis and EPOsynthesis; and in alternative embodiments chimeric proteins of theinvention comprise: one or more (a plurality of, multiple) proteinDNA-binding domains, e.g., zinc finger binding domains, specific for thepromoter region of an erythropoietin (EPO) gene or an ErythropoietinStimulating Protein (NESP); one or more consensus nuclear localizationproteins; one or more cell-penetrating peptides; and, one or moretranscription activation domain. Erythropoietin (EPO) and ErythropoietinStimulating Protein (NESP) polypeptides and genes encoding them are wellknown in the art. For example, see U.S. Pat. Nos. 7,262,166, and6,586,398, describing the Erythropoietin Stimulating Protein (NESP)protein and gene.

In alternative embodiments, polypeptides and/or peptides used topractice the invention comprise a recombinant protein, a syntheticprotein, a peptidomimetic, a non-natural peptide, or a combinationthereof. Peptides and proteins used to practice the invention can berecombinantly expressed in vitro or in vivo. The peptides andpolypeptides used to practice the invention can be made and isolatedusing any method known in the art. Polypeptide and peptides used topractice the invention can also be synthesized, whole or in part, usingchemical methods well known in the art. See e.g., Caruthers (1980)Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res.Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins,Formulation, Processing and Delivery Systems (1995) Technomic PublishingCo., Lancaster, Pa. For example, peptide synthesis can be performedusing various solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) including anyautomated polypeptide synthesis process known in the art.

The chimeric peptides and polypeptides used to practice the inventioncan also be glycosylated. The glycosylation can be addedpost-translationally either chemically or by cellular biosyntheticmechanisms, wherein the later incorporates the use of knownglycosylation motifs, which can be native to the sequence or can beadded as a peptide or added in the nucleic acid coding sequence. Theglycosylation can be O-linked or N-linked.

In alternative embodiments, compositions of the invention, including thechimeric peptides and polypeptides used to practice the invention, cancomprise an oligopeptide, peptide, polypeptide, or protein sequence, orto a fragment, portion, or subunit of any of these and to naturallyoccurring or synthetic molecules, including, e.g., peptidomimetics andnon-natural amino acids. In alternative embodiments, chimeric peptidesand polypeptides used to practice the invention comprise amino acidsjoined to each other by peptide bonds or modified peptide bonds and maycomprise modified amino acids other than the 20 gene-encoded aminoacids. The chimeric peptides and polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Modificationscan be designed anywhere in the polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.The same type of modification can be made in the same or varying degreesat several sites in a given chimeric polypeptide.

In alternative embodiments, chimeric peptides and polypeptides used topractice the invention can have many types of modifications, e.g.,modifications including acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof a phosphatidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pegylation,phosphorylation, prenylation, racemization, selenoylation, sulfation andtransfer-RNA mediated addition of amino acids to protein such asarginylation. See for example, Creighton, T. E., Proteins—Structure andMolecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp. 1-12 (1983)). In another embodiment, achimeric polypeptide of the invention (a DRP) can be glycol-pegylated asdescribed in U.S. Pat. No. 7,405,198; or can be glycosylated asdescribed in U.S. Pat. Nos. 7,276,475 or 7,399,613, or 7,338,933, thelater describing O-linked glycosylation of peptides. In one embodiment,a chimeric polypeptide of the invention (a DRP) used to practice thisinvention can be acylated as described e.g., in U.S. Pat. No. 7,273,921.In another embodiment, a chimeric polypeptide of the invention (a DRP)can be pegylated (a process that can be called “PEGylation”) asdescribed e.g., in U.S. Patent App. Pub. No. 20030166566 and20060264377.

In alternative embodiments, chimeric peptides and polypeptides used topractice the invention can comprise any “mimetic” and/or“peptidomimetic” form. In alternative embodiments, peptides andpolypeptides used to practice the invention can comprise syntheticchemical compounds which have substantially the same structural and/orfunctional characteristics of natural polypeptides. A mimetic used topractice the invention can be either entirely composed of synthetic,non-natural analogues of amino acids, or, is a chimeric molecule ofpartly natural peptide amino acids and partly non-natural analogs ofamino acids. A mimetic used to practice the invention can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or activity.

Routine experimentation will determine whether a synthetic molecule ormimetic is effective for practicing the invention, e.g., has EPOpromoter-specific binding activity, e.g., an EPO-specific zinc fingerDNA-binding activity, or a nuclear localization peptide activity, or acell-penetrating peptide activity, or a transcription activation (TA)peptide domain and/or a transcription repression peptide activity.Methodologies detailed herein and others known to persons skilled in theart may be used to select or guide one to choose effective mimetic forpracticing the compositions and/or methods of this invention.

Polypeptide mimetic compositions for practicing the invention cancomprise any combination of non-natural structural components. Inalternative aspects, mimetic compositions for practicing the inventioncan comprise one or all of the following three structural groups: a)residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. For example, a polypeptide can be characterized as a mimetic whenall or some of its residues are joined by chemical means other thannatural peptide bonds. Individual peptidomimetic residues can be joinedby peptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand

Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide Backbone Modifications,” Marcell Dekker, NY). A polypeptide canalso be characterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues.Non-natural residues are well described in the scientific and patentliterature; a few exemplary non-natural compositions useful as mimeticsof natural amino acid residues and guidelines are described below.Mimetics of aromatic amino acids can be generated by replacing by, e.g.,D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D-or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids used to practice this invention can begenerated by substitution by, e.g., non-carboxylate amino acids whilemaintaining a negative charge; (phosphono)alanine; sulfated threonine.Carboxyl side groups (e.g., aspartyl or glutamyl) can also beselectively modified by reaction with carbodiimides (R′—N—C—N—R′) suchas, e.g., 1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,e.g., under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics that can be used include, e.g., those generatedby hydroxylation of proline and lysine; phosphorylation of the hydroxylgroups of seryl or threonyl residues; methylation of the alpha-aminogroups of lysine, arginine and histidine; acetylation of the N-terminalamine; methylation of main chain amide residues or substitution withN-methyl amino acids; or amidation of C-terminal carboxyl groups.

Polypeptides used to practice this invention can comprise anyheterologous sequence, e.g., a “tag” for identifying and/or isolating achimeric polypeptide of the invention, and/or a signal sequence (aleader sequence) e.g., for secreting a recombinant antibody orinhibitory polypeptide used to practice the invention from a cell, e.g.,a production host cell.

Generating and Manipulating Nucleic Acids

In alternative aspects, because the chimeric polypeptides used topractice this invention can be recombinantly generated, and can be usedin recombinant form, the invention provides nucleic acids, whichthemselves can be recombinant, to make them. In alternative embodiments,nucleic acids of the invention are made, isolated and/or manipulated by,e.g., cloning and expression of cDNA libraries, amplification of messageor genomic DNA by PCR, and the like.

The nucleic acids used to practice this invention, whether RNA,antisense nucleic acid, cDNA, genomic DNA, vectors, plasmids, viruses orhybrids thereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., the chimeric proteins used to practicethis invention) generated from these nucleic acids can be individuallyisolated or cloned and tested for a desired activity. Any recombinantexpression system can be used, including e.g. bacterial, fungal,mammalian, yeast, insect or plant cell expression systems.

Alternatively, nucleic acids used to practice this invention can besynthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrook,ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, ColdSpring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N. Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

The invention provides and uses chimeric proteins (also called “fusionproteins”) and nucleic acids encoding them. Any polypeptide used topractice this invention (e.g., chimeric protein) can be fused to aheterologous peptide or polypeptide, such as a peptide for targeting thepolypeptide to a desired cell type, e.g., any cell involved inerythropoiesis. In alternative embodiments, a heterologous peptide orpolypeptide joined or fused to a protein used to practice this inventioncan be an N-terminal identification peptide which imparts a desiredcharacteristic, such as fluorescent detection, increased stabilityand/or simplified purification. Peptides and polypeptides used topractice this invention can also be synthesized and expressed as fusionproteins with one or more additional domains linked thereto for, e.g.,producing a more immunogenic peptide, to more readily isolate arecombinantly synthesized peptide, and the like. Detection andpurification facilitating domains include, e.g., metal chelatingpeptides such as polyhistidine tracts and histidine-tryptophan modulesthat allow purification on immobilized metals, protein A domains thatallow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Nucleic acids used to practice this invention can be or comprise anoligonucleotide, nucleotide, polynucleotide, or a fragment of any ofthese, or DNA or RNA of genomic or synthetic origin, which may besingle-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material, natural or synthetic in origin. Compounds used topractice this invention include DNA or RNA (e.g., mRNA, rRNA, tRNA,iRNA) of genomic or synthetic origin which may be single-stranded ordouble-stranded; and can be a sense or antisense strand, or a peptidenucleic acid (PNA), or any DNA-like or RNA-like material, natural orsynthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g.,e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practicethis invention include nucleic acids, i.e., oligonucleotides, containingknown analogues of natural nucleotides. Compounds use to practice thisinvention include nucleic-acid-like structures with synthetic backbones,see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practicethis invention include “oligonucleotides” including a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandsthat may be chemically synthesized. Compounds use to practice thisinvention include synthetic oligonucleotides having no 5′ phosphate, andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

In alternative aspects, compounds used to practice this inventioninclude genes or any segment of DNA involved in producing a polypeptidechain (e.g., a chimeric protein of this invention); it can includeregions preceding and following the coding region (leader and trailer)as well as, where applicable, intervening sequences (introns) betweenindividual coding segments (exons). “Operably linked” can refer to afunctional relationship between two or more nucleic acid (e.g., DNA)segments, e.g., an EPO specific protein and the EPO coding sequence,where a chimeric protein of the invention activates or upregulates thepromoter to activate or upregulate transcription of the EPO codingsequence transcript; e.g., by binding to an EPO gene promoter. EPO genepromoters are well known in the art, see e.g., Tsuchiya (1997) J.Biochem. 121(2):193-196; Tarumoto (2000) Blood 96(5):1716-17122. EPOgene promoters have been cloned and characterized, e.g., in U.S. PatentApp. Pub. No. 20080312179. Erythropoietin stimulating protein genes andpromoters are also described e.g., in U.S. Patent App. Pub. Nos.20060264377 and 20030166566.

In alternative aspects, it can refer to the functional relationship oftranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter can be operably linked to a coding sequence, such asa nucleic acid used to practice this invention, if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. In alternative aspects, promotertranscriptional regulatory sequences can be operably linked to atranscribed sequence where they can be physically contiguous to thetranscribed sequence, i.e., they can be cis-acting. In alternativeaspects, transcriptional regulatory sequences, such as enhancers, neednot be physically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

In alternative aspects, the invention comprises use of “expressioncassettes” comprising a nucleotide sequence used to practice thisinvention, which can be capable of affecting expression of the nucleicacid, e.g., a structural gene or a transcript (e.g., encoding a chimericpolypeptide of this invention) in a host cell compatible with suchsequences. Expression cassettes can include at least a promoter operablylinked with the polypeptide coding sequence or inhibitory sequence; and,in one aspect, with other sequences, e.g., transcription terminationsignals. Additional factors necessary or helpful in effecting expressionmay also be used, e.g., enhancers.

In alternative aspects, expression cassettes used to practice thisinvention also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like. Inalternative aspects, a “vector” used to practice this invention cancomprise a nucleic acid (e.g., encoding a chimeric protein of thisinvention) that can infect, transfect, transiently or permanentlytransduce a cell. In alternative aspects, a vector used to practice thisinvention (e.g., comprising coding sequence for a chimeric protein ofthis invention) can be a naked nucleic acid, or a nucleic acid complexedwith protein or lipid. In alternative aspects, vectors used to practicethis invention can comprise viral or bacterial nucleic acids and/orproteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). In alternative aspects, vectors used to practice thisinvention can include, but are not limited to replicons (e.g., RNAreplicons, bacteriophages) to which fragments of DNA may be attached andbecome replicated. Vectors thus include, but are not limited to RNA,autonomous self-replicating circular or linear DNA or RNA (e.g.,plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879),and can include both the expression and non-expression plasmids. Inalternative aspects, the vector used to practice this invention can bestably replicated by the cells during mitosis as an autonomousstructure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” used to practice this inventioninclude all sequences capable of driving transcription of a codingsequence in a cell, e.g., a mammalian cell such as a human cell. Thus,promoters used in the constructs of the invention include cis-actingtranscriptional control elements and regulatory sequences that areinvolved in regulating or modulating the timing and/or rate oftranscription of a sequence encoding a chimeric protein of thisinvention. For example, a promoter used to practice this invention canbe a cis-acting transcriptional control element, including an enhancer,a promoter, a transcription terminator, an origin of replication, achromosomal integration sequence, 5′ and 3′ untranslated regions, or anintronic sequence, which are involved in transcriptional regulation.These cis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription.

“Constitutive” promoters used to practice this invention can be thosethat drive expression continuously under most environmental conditionsand states of development or cell differentiation. “Inducible” or“regulatable” promoters used to practice this invention can directexpression of the nucleic acid of the invention under the influence ofenvironmental conditions, specific physiologic or pathologic conditionsand/or developmental conditions.

Kits and Instructions

The invention provides kits comprising compositions and methods of theinvention, including instructions for use thereof. As such, kits, cells,vectors and the like can also be provided.

For example, in alternative embodiments, the invention provides kitscomprising compositions comprising e.g., a set of (e.g., the pluralityof) chimeric proteins of the invention; or a liquid or aqueousformulation of the invention; or a vesicle, liposome, nanoparticle ornanolipid particle of the invention. In one aspect, the kit furthercomprising instructions for practicing any composition or method of theinvention, e.g., in vitro or ex vivo methods for directing in vitro, exvivo or ex vivo synthesis of EPO in a mammalian cell.

Formulations

In alternative embodiments, the invention provides compositions for usein in vitro, ex vivo or in vivo methods (including methods of theinvention) for generating EPO message (transcripts) and/or proteins;including re-programming a mammalian cell to generate new or more EPOtranscript or protein. In alternative embodiments, these compositionscomprise a plurality of (a set of) proteins and/or nucleic acidsformulated for these purposes, e.g., one or a plurality of chimericproteins of the invention, and/or nucleic acids (e.g., vectors) thatencode them, formulated in a buffer, in a saline solution, in a powder,an emulsion, in a vesicle, in a liposome, in a nanoparticle, in ananolipoparticle and the like.

In alternative embodiments, the compositions can be formulated in anyway and can be applied in a variety of concentrations and formsdepending on the desired in vitro, ex vivo or in vivo conditions, adesired in vitro, ex vivo or in vivo method of administration and thelike. Details on techniques for in vitro, ex vivo or in vivoformulations and administrations are well described in the scientificand patent literature.

Alternative formulations and/or carriers of the chimeric proteins ornucleic acids used to practice this invention are well known in the art.Formulations and/or carriers used to practice this invention can be informs such as tablets, pills, powders, capsules, liquids, gels, syrups,slurries, suspensions, etc., suitable for in vitro or ex vivoapplications.

In alternative embodiments chimeric proteins or nucleic acids used topractice this invention are in admixture with an aqueous and/or buffersolution or as an aqueous and/or buffered suspension, e.g., including asuspending agent, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate. Formulations can be adjusted for osmolarity, e.g., byuse of an appropriate buffer.

For example, chimeric proteins of the invention can be formulated insustained-release pharmaceutical formulations comprising carboxymethylether cellulose polymer, as described e.g., in U.S. Pat. No. 7,282,480.In one aspect, the sodium carboxymethyl ether cellulose is in aconcentration range of about 0.5% to about 7% total formula weight. Thispharmaceutical formulation can be administered e.g., thrice per twoweeks, once per week, once per two weeks, once per three weeks, once permonth, once per five weeks, or once per six weeks; e.g., at about 1μg/ml to about 2000 μg/ml protein per formulation.

In alternative embodiments, oil-based formulations are used for invitro, ex vivo or in vivo application of the compositions of theinvention. Oil-based suspensions can be formulated by suspending the setof chimeric DRP proteins of the invention in a vegetable oil, such asarachis oil, olive oil, sesame oil or coconut oil, or in a mineral oilsuch as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No.5,716,928 describing using essential oils or essential oil componentsfor increasing bioavailability and reducing inter- and intra-individualvariability of hydrophobic compounds; see also U.S. Pat. No. 5,858,401.These formulations can be preserved by the addition of an antioxidantsuch as ascorbic acid. The formulations of the invention can also be inthe form of oil-in-water emulsions. The oily phase can be a vegetableoil or a mineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. Formulations can also contain a buffer,preservative or a coloring agent.

In practicing this invention, the compounds (e.g., formulations) of theinvention can comprise a solution of chimeric proteins or nucleic acidsused to practice this invention dissolved in a pharmaceuticallyacceptable carrier, e.g., acceptable vehicles and solvents that can beemployed include water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any fixed oil can be employedincluding synthetic mono- or diglycerides, or fatty acids such as oleicacid. In one embodiment, solutions and formulations used to practice theinvention are sterile and can be manufactured to be generally free ofundesirable matter. In one embodiment, these solutions and formulationsare sterilized by conventional, well known sterilization techniques.

The solutions and formulations used to practice the invention cancomprise auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents, e.g., sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent (e.g., chimeric proteins) in these formulations can varywidely, and can be selected primarily based on fluid volumes,viscosities and the like, in accordance with the particular mode of invitro, ex vivo or in vivo administration selected and the desiredresults, e.g., for de-differentiating or re-programming a mammaliancell.

The solutions and formulations used to practice the invention can belyophilized; for example, the invention provides a stable lyophilizedformulation comprising a chimeric protein or nucleic acid of theinvention. In one aspect, this formulation is made by lyophilizing asolution comprising a chimeric protein or nucleic acid of the inventionand a bulking agent, e.g., mannitol, trehalose, raffinose, and sucroseor mixtures thereof. A process for preparing a stable lyophilizedformulation can include lyophilizing a solution about 2.5 mg/mL protein,about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate bufferhaving a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patentapp. no. 20040028670.

The compositions and formulations of the invention can be delivered bythe use of liposomes (see also discussion, below). By using liposomes,particularly where the liposome surface carries ligands specific fortarget cells, or are otherwise preferentially directed to a specifictissue or organ type, one can focus the delivery of the active agentinto a target cells in an in vitro or ex vivo application.

Nanoparticles, Nanolipoparticles and Liposomes

The invention also provides nanoparticles, nanolipoparticles, vesiclesand liposomal membranes comprising compounds used to practice themethods and compositions, e.g., a chimeric protein or nucleic acid ofthe invention, e.g., to deliver compositions of the invention tomammalian cells in vitro, ex vivo or in vivo. In alternativeembodiments, these compositions are designed to target specificmolecules, including biologic molecules, such as polypeptides, includingcell surface polypeptides, e.g., for targeting a desired cell type,e.g., a mammalian cell targeted for de-differentiation orre-programming.

The invention provides multilayered liposomes comprising compounds usedto practice this invention, e.g., as described in Park, et al., U.S.Pat. Pub. No. 20070082042. The multilayered liposomes can be preparedusing a mixture of oil-phase components comprising squalane, sterols,ceramides, neutral lipids or oils, fatty acids and lecithins, to about200 to 5000 nm in particle size, to entrap a composition of thisinvention (e.g., a chimeric protein or nucleic acid of the invention).

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating an active agent (e.g., chimeric protein ornucleic acid of the invention), the method comprising providing anaqueous solution in a first reservoir; providing an organic lipidsolution in a second reservoir, and then mixing the aqueous solutionwith the organic lipid solution in a first mixing region to produce aliposome solution, where the organic lipid solution mixes with theaqueous solution to substantially instantaneously produce a liposomeencapsulating the active agent; and immediately then mixing the liposomesolution with a buffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions used to practice this inventioncomprise a substituted ammonium and/or polyanions, e.g., for targetingdelivery of a compound (e.g., chimeric protein or nucleic acid) used topractice this invention to a desired cell type, as described e.g., inU.S. Pat. Pub. No. 20070110798.

The invention also provides nanoparticles comprising compounds (e.g.,chimeric protein or nucleic acid) used to practice this invention in theform of active agent-containing nanoparticles (e.g., a secondarynanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. Inone embodiment, the invention provides nanoparticles comprising afat-soluble active agent of this invention or a fat-solubilizedwater-soluble active agent to act with a bivalent or trivalent metalsalt.

In one embodiment, solid lipid suspensions can be used to formulate andto deliver compositions of the invention to mammalian cells in vitro, exvivo or in vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.

Polypeptide In Vitro, Ex Vivo or In Vivo Delivery Vehicles

In alternative embodiments, any delivery vehicle can be used to practicethe methods or compositions of this invention, e.g., to deliver chimericproteins of the invention to a host cell, e.g., a mammalian cell, invitro, ex vivo or in vivo. For example, delivery vehicles comprisingpolycations, cationic polymers and/or cationic peptides, such aspolyethyleneimine derivatives, can be used e.g. as described, e.g., inU.S. Pat. Pub. No. 20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used toformulate a composition of the invention, wherein a surfactant isassociated with a chimeric proteins of the invention via a noncovalentbond e.g. as described, e.g., in U.S. Pat. Pub. No. 20040151766.

In one embodiment, a covalent conjugate between a poly(alkylene oxide)and a glycosylated or non-glycosylated chimeric protein of the inventionis used, where a poly(alkylene oxide) can be conjugated to a chimericprotein via a glycosyl linking group, and a glycosyl linking group canbe interposed between a chimeric protein and a poly(alkylene oxide). Acovalent conjugate can be formed by contacting a chimeric protein with aglycosyltransferase and a modified sugar donor; the glycosyltransferasetransfers the modified sugar moiety to the chimeric protein to form acovalent conjugate; the modified sugar moiety can be a poly(alkyleneoxide). See e.g., U.S. Pat. No. 7,416,858.

In one embodiment, a chimeric protein used to practice this inventioncan be applied to cells as polymeric hydrogels or water-solublecopolymers, e.g., as described in U.S. Pat. No. 7,413,739; for example,a chimeric protein can be polymerized through a reaction between astrong nucleophile and a conjugated unsaturated bond or a conjugatedunsaturated group, by nucleophilic addition, wherein each precursorcomponent comprises at least two strong nucleophiles or at least twoconjugated unsaturated bonds or conjugated unsaturated groups. In oneembodiment, a chimeric protein used to practice this invention can beapplied to cells using vehicles with cell membrane-permeant peptideconjugates, e.g., as described in U.S. Pat. Nos. 7,306,783; 6,589,503.In one aspect, the chimeric protein itself is conjugated to a cellmembrane-permeant peptide. In one embodiment, a chimeric protein and/orthe delivery vehicle are conjugated to a transport-mediating peptide,e.g., as described in U.S. Pat. No. 5,846,743, describingtransport-mediating peptides that are highly basic and bind topoly-phosphoinositides.

In one embodiment, electro-permeabilization is used as a primary oradjunctive means to deliver a chimeric protein of the invention to acell, e.g., using any electroporation system as described e.g. in U.S.Pat. Nos. 7,109,034; 6,261,815; 5,874,268.

Pharmaceutical Compositions

The invention provides compositions for ameliorating anemia and/orstimulating EPO synthesis and EPO synthesis, including pharmaceuticalcompositions, e.g., in the manufacture of medicaments for amelioratinganemia and/or stimulating erythropoiesis and EPO synthesis.

In alternative embodiments, the compositions (e.g., comprising chimericproteins and/or nucleic acids encoding chimeric proteins) of theinvention are formulated with a pharmaceutically acceptable carrier. Inalternative embodiments, the pharmaceutical compositions of theinvention can be administered subcutaneously, parenterally, topically,orally or by local administration, such as by aerosol or transdermally.The pharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition (e.g., anemia) or disease and the degree of illness, thegeneral medical condition of each patient, the resulting preferredmethod of administration and the like. Details on techniques forformulation and administration are well described in the scientific andpatent literature, see, e.g., the latest edition of Remington'sPharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

Therapeutic agents of the invention can be administered alone or as acomponent of a pharmaceutical formulation (composition). The compoundsmay be formulated for administration in any convenient way for use inhuman or veterinary medicine. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Formulations of the compositions (e.g., chimeric proteins) of theinvention include those suitable for subcutaneous, spray, nebulized,oral/nasal, topical, parenteral, rectal, and/or intravaginaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single or multiple dosage form will varydepending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single or multiple dosage form willgenerally be that amount of the compound which produces a therapeuticeffect.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for liquid or oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in appropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, e.g., push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and a coatingsuch as glycerol or sorbitol. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., a chimericpolypeptide or peptidomimetic of the invention) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil-based pharmaceuticals are particularly useful for administration ofthe compositions (e.g., chimeric proteins) of the invention. Oil-basedsuspensions can be formulated by suspending an active agent (e.g.,chimeric protein of the invention) in a vegetable oil, such as arachisoil, olive oil, sesame oil or coconut oil, or in a mineral oil such asliquid paraffin; or a mixture of these. See e.g., U.S. Pat. No.5,716,928 describing using essential oils or essential oil componentsfor increasing bioavailability and reducing inter- and intra-individualvariability of orally administered hydrophobic pharmaceutical compounds(see also U.S. Pat. No. 5,858,401). The oil suspensions can contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol or sucrose. These formulations can bepreserved by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto (1997) J. Pharmacol.Exp. Ther. 281:93-102. The pharmaceutical formulations of the inventioncan also be in the form of oil-in-water emulsions. The oily phase can bea vegetable oil or a mineral oil, described above, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

In practicing this invention, the pharmaceutical compounds can also beadministered by in intranasal, intraocular and intravaginal routesincluding suppositories, insufflation, powders and aerosol formulations(for examples of steroid inhalants, see Rohatagi (1995) J. Clin.Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.75:107-111). Suppositories formulations can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at body temperatures and will therefore melt inthe body to release the drug. Such materials are cocoa butter andpolyethylene glycols.

In practicing this invention, the pharmaceutical compounds can bedelivered by transdermally, by a topical route, and/or can be formulatedas applicator sticks, solutions, suspensions, emulsions, gels, creams,ointments, pastes, jellies, paints, powders, and aerosols. Inalternative embodiments, topical formulations of the inventioncomprising e.g., chimeric proteins of the invention and/or nucleic acidsencoding them, comprise an ointment, a cream, a powder, an emulsion, agel, a glycerogelatin (a preparation made from glycerin and glycerinatedgelatin), a paste, a plaster, a sprayable composition or a lotion.

In alternative embodiments, pharmaceutical compositions of the inventionare formulated as cream formulation, e.g., containing no or up to 12%urea, e.g., comprising aqua, caprylic and/or capric triglyceride,pentylene glycol, hydrogenated lecithin, butyrospermum parkii, glycerin,squalane, ceramide 3 and the like. In alternative embodiments,pharmaceutical compositions of the invention are formulated as waterbased or non-water based suspensions or pastes. In alternativeembodiments, pharmaceutical compositions of the invention are formulatedmoist or dry, and can be combined with adjuvants and/or form ointments,creams, powders, emulsions, lotions, gels, pastes, plasters,glycerogelatiness or sprays. In alternative embodiments, ointments inthe form of semisolid preparation are used for external applications,e.g., to skin or mucous membranes. In alternative embodiments, ointmentscan be prepared by incorporation or by fusion; or the ointments cancomprise hydrocarbon bases, e. g. petrolatum USP, white petrolatum USP,yellow ointment USP, or white ointment USP, absorption bases, e. g.hydrophilic petrolatum USP, or lanolin USP, water-removable bases, e. g.hydrophilic ointment USP, water-soluble bases, e. g. a polyethyleneglycol ointment.

In alternative embodiments, creams are formulated as semisolidpreparations, and compositions of the invention can be dissolved ordispensed in either an oil-in-water emulsion or in another type ofwater-washable base. Creams also can be used rectally and/or vaginally.Gels are semisolid systems consisting of dispersions of large moleculesin an aqueous liquid vehicle rendered jelly-like through the addition ofa gelling agent. Examples of gelling agents are carbomer 934, cellulosederivatives as carboxymethylcellulose or hydroxypropylmethyl-cellulose,and natural gums as tragacanth. Carbomers are high molecular weightwater-soluble polymers of acrylic acid cross-linked with allyl ethers ofsucrose and/or pentaerythritol.

In alternative embodiments, pastes are formulated as semisolidpreparations for application to the skin or mucous membranes. Pastes maycomprise zinc oxide, starch and/or white petrolatum. In alternativeembodiments, plasters are formulated as solid or semi-solid adhesivemasses spread upon a backing material of paper, fabric, moleskin orplastic. The adhesive material can be a rubber base or synthetic resin.Plasters can be applied to skin to provide prolonged contact at thesite. In alternative embodiments, glycerogelatins comprise gelatin,glycerol and water.

In alternative embodiments, sprays are formulated as aqueous oroleaginous solutions in the form of coarse droplets or as finely dividedsolids to be applied topically, most usually to the nasal-pharyngealtract or to the skin. Any mechanical device for spraying or nebulizingcan be used in practicing the invention.

In practicing this invention, the pharmaceutical compounds also can bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of drug whichslowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym.Ed. 7:623-645; as biodegradable and injectable gel formulations, see,e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres fororal administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674.

In practicing this invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs.

For IV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

Pharmaceutical compositions of this invention can be formulated in aliquid form, and can be injected intravenously or subcutaneously. In oneembodiment, a composition of this invention, e.g., comprising a chimericpolypeptide of this invention, is administered with EPO.

Pharmaceutical compositions of this invention can be formulated asstable liquid preparations, e.g., as described in U.S. Pat. App. Pub.No. 20080039371, where the formulation would comprise a chimericpolypeptide of this invention and amino acids, e.g., leucine,isoleucine, threonine, glutamic acid, aspartic acid and/or phenylalanine or any combination thereof; where in one embodiment theformulation is free of preservatives, urea and HAS. In alternativeembodiments the formulation has an osmolality about 200 to 400mosmol/kg, or 250 to 300 mosmol/kg, or 260 to 290 mosmol/kg.Pharmaceutical compositions of this invention can be formulated asdescribed e.g. in U.S. Pat. App. Pub. No. 20070293419, using e.g., anon-ionic surfactant, polyhydric alcohol, neutral amino acid and sugaralcohol as stabilizers; isotonic reagent; and/or a buffering reagent.Pharmaceutical compositions of this invention can be formulated asdescribed e.g. in U.S. Pat. App. Pub. No. 20070128231, using e.g., a pHbuffering agent a sodium phosphate buffer; as a stabilizertris-(hydroxymethyl)-aminomethane; and/or a pharmaceutically effectivequantity of a chimeric polypeptide or nucleic acid of this invention.

The compositions and formulations of the invention can be delivered bythe use of liposomes (see also discussion, below). By using liposomes,particularly where the liposome surface carries ligands specific fortarget cells, or are otherwise preferentially directed to a specificorgan, one can focus the delivery of the active agent into target cellsin vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed(1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin.Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In therapeutic applications, compositionsare administered to a subject already suffering from a condition,infection or disease in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the condition, infectionor disease and its complications (a “therapeutically effective amount”).For example, in alternative embodiments, pharmaceutical compositions ofthe invention are administered in an amount sufficient to treat, preventand/or ameliorate anemia or less than normal levels of erythropoiesis.The amount of pharmaceutical composition adequate to accomplish this isdefined as a “therapeutically effective dose.” The dosage schedule andamounts effective for this use, i.e., the “dosing regimen,” will dependupon a variety of factors, including the stage of the disease orcondition, the severity of the disease or condition, the general stateof the patient's health, the patient's physical status, age and thelike. In calculating the dosage regimen for a patient, the mode ofadministration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra). The state of the art allows theclinician to determine the dosage regimen for each individual patient,active agent and disease or condition treated. Guidelines provided forsimilar compositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods of the invention are correct andappropriate. For example, in one embodiment a therapeutically effectiveamount of a chimeric polypeptide of this invention is a dosage ofbetween about 0.025 to 0.5 milligram per 1 kilogram of body weight ofthe patient; or, a therapeutically effective amount is a dosage ofbetween about 0.025 to 0.2 milligram, or 0.05 to 0.1 milligram, or 0.075to 0.5 milligram, or 0.2 to 0.4 milligram, of the compound per 1kilogram of body weight of the patient.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required and tolerated by thepatient. The formulations should provide a sufficient quantity of activeagent to effectively treat, prevent or ameliorate a conditions, diseasesor symptoms as described herein. For example, an exemplarypharmaceutical formulation for oral administration can be in a dailyamount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or moreμg per kilogram of body weight per day of chimeric protein. In analternative embodiment, dosages are from about 1 mg to about 4 mg per kgof body weight per patient per day of chimeric protein are used. Lowerdosages can be used, in contrast to administration by inhalation (e.g.,bys spray or nebulizer), orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in subcutaneous, topical or oral administration or administering bynebulizers, powders, spray or inhalation. Actual methods for preparingparenterally or non-parenterally administrable formulations will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's, supra.

The methods of the invention can further comprise co-administration withother drugs or pharmaceuticals, e.g., compositions for treating anemiaand related symptoms or conditions. For example, the methods and/orcompositions and formulations of the invention can be co-formulated withand/or co-administered with antibiotics (e.g., antibacterial orbacteriostatic peptides or proteins), including those effective againstgram negative bacteria, fluids, cytokines, immunoregulatory agents,anti-inflammatory agents, complement activating agents, such as peptidesor proteins comprising collagen-like domains or fibrinogen-like domains(e.g., a ficolin), carbohydrate-binding domains, and the like andcombinations thereof.

Nanoparticles and Liposomes

The invention also provides nanoparticles and liposomal membranescomprising compounds of this invention which target specific molecules,including biologic molecules, such as polypeptide, including cellsurface polypeptides. Thus, in alternative embodiments, the inventionprovides nanoparticles and liposomal membranes targeting EPO-producingcells, or any cell comprising an EPO protein coding sequence (which asdescribed above can be a heterologous EPO protein coding sequence.

In alternative embodiments, the invention provides nanoparticles andliposomal membranes comprising (in addition to comprising compounds ofthis invention) molecules, e.g., peptides or antibodies, thatselectively target EPO-producing cells or any cell comprising aheterologous EPO protein coding sequence. In alternative embodiments,the invention provides nanoparticles and liposomal membranes usingreceptors to target EPO-producing cells or any cell comprising aheterologous EPO protein coding sequence.

Thus, in one aspect, the compositions of the invention are specificallytargeted for inhibiting, ameliorating and/or preventing anemia, and/orincreasing levels of EPO produced by a cell and/or individual.

The invention also provides nanocells to allow the sequential deliveryof two different therapeutic agents with different modes of action ordifferent pharmacokinetics, at least one of which comprises acomposition of this invention. A nanocell is formed by encapsulating ananocore with a first agent inside a lipid vesicle containing a secondagent; see, e.g., Sengupta, et al., U.S. Pat. Pub. No. 20050266067. Theagent in the outer lipid compartment is released first and may exert itseffect before the agent in the nanocore is released. The nanocelldelivery system may be formulated in any pharmaceutical composition fordelivery to patients suffering from a diseases or condition related toanemia. In treating cancer, a traditional antineoplastic agent iscontained in the outer lipid vesicle of the nanocell, and an agent ofthis invention is loaded into the nanocore.

The invention also provides multilayered liposomes comprising compoundsof this invention, e.g., for transdermal absorption, e.g., as describedin Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayeredliposomes can be prepared using a mixture of oil-phase componentscomprising squalane, sterols, ceramides, neutral lipids or oils, fattyacids and lecithins, to about 200 to 5000 nm in particle size, to entrapa composition of this invention.

A multilayered liposome of the invention may further include anantiseptic, an antioxidant, a stabilizer, a thickener, and the like toimprove stability. Synthetic and natural antiseptics can be used, e.g.,in an amount of 0.01% to 20%. Antioxidants can be used, e.g., BHT,erysorbate, tocopherol, astaxanthin, vegetable flavonoid, andderivatives thereof, or a plant-derived antioxidizing substance. Astabilizer can be used to stabilize liposome structure, e.g., polyolsand sugars. Exemplary polyols include butylene glycol, polyethyleneglycol, propylene glycol, dipropylene glycol and ethyl carbitol;examples of sugars are trehalose, sucrose, mannitol, sorbitol andchitosan, or monosaccharides or oligosaccharides, or a high molecularweight starch. A thickener can be used for improving the dispersionstability of constructed liposomes in water, e.g., a natural thickeneror an acrylamide, or a synthetic polymeric thickener. Exemplarythickeners include natural polymers, such as acacia gum, xanthan gum,gellan gum, locust bean gum and starch, cellulose derivatives, such ashydroxy ethylcellulose, hydroxypropyl cellulose and carboxymethylcellulose, synthetic polymers, such as polyacrylic acid, poly-acrylamideor polyvinylpyrollidone and polyvinylalcohol, and copolymers thereof orcross-linked materials.

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating a therapeutic product comprising providing anaqueous solution in a first reservoir; providing an organic lipidsolution in a second reservoir, wherein one of the aqueous solution andthe organic lipid solution includes a therapeutic product; mixing theaqueous solution with said organic lipid solution in a first mixingregion to produce a liposome solution, wherein the organic lipidsolution mixes with said aqueous solution so as to substantiallyinstantaneously produce a liposome encapsulating the therapeuticproduct; and immediately thereafter mixing the liposome solution with abuffer solution to produce a diluted liposome solution. The inventionalso provides nanoparticles comprising compounds of this invention todeliver a composition of the invention as a drug-containingnanoparticles (e.g., a secondary nanoparticle), as described, e.g., inU.S. Pat. Pub. No. 20070077286. In one embodiment, the inventionprovides nanoparticles comprising a fat-soluble drug of this inventionor a fat-solubilized water-soluble drug to act with a bivalent ortrivalent metal salt.

Products of Manufacture, Implants and Artificial Organs

The invention also provides products of manufacture comprising cells ofthe invention (e.g., cells comprising coding sequence for chimericpolypeptides of this invention), and use of cells made by methods ofthis invention, including for example implants and artificial organs,bioreactor systems, cell culture systems, plates, dishes, tubes, bottlesand flasks comprising cells of this invention. Any implant, artificialorgan, bioreactor systems, cell culture system, cell culture plate, dish(e.g., petri dish), cell culture tube and/or cell culture flask (e.g., aroller bottle) can be used to practice this invention.

In alternative embodiments the invention provides a bioreactor, implant,stent, artificial organ or similar device comprising a cell of theinvention, or cells made by a method of this invention; for example,including implants as described in U.S. Pat. Nos. 7,388,042; 7,381,418;7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; and U.S. Pat.App. Pub. Nos. 20040127987; 20080119909 (describing auricular implants);20080118549 (describing ocular implants); 20080020015 (describing abioactive wound dressing); 20070254005 (describing heart valvebio-prostheses, vascular grafts, meniscus implants); 20070059335;20060128015 (describing liver implants).

Implanting Cells In Vivo

In alternative embodiments, the methods of the invention also compriseimplanting or engrafting cells of the invention (e.g., cells comprisingcoding sequence for chimeric polypeptides of this invention) in avessel, tissue or organ; and in one aspect, comprise implanting orengrafting these cells in a vessel, tissue or organ ex vivo or in vivo,or implanting or engrafting these cells in an individual in needthereof.

Cells can be removed from an individual, treated using the compositionsand/or methods of this invention, and reinserted (e.g., injected orengrafted) into a tissue, organ or into the individual, using any knowntechnique or protocol. For example, de-differentiated re-programmedcells, or re-programmed differentiated cells, can be re-implanted (e.g.,injected or engrafted) using microspheres e.g., as described in U.S.Pat. No. 7,442,389; e.g., in one aspect, the cell carrier comprises abulking agent comprising a plurality of round and smoothpolymethylmethacrylate microparticles preloaded within a mixing anddelivery system and an autologous carrier comprising these cells. Inanother embodiment, the cells are readministered to a tissue, an organand/or an individual in need thereof in a biocompatible crosslinkedmatrix, as described e.g., in U.S. Pat. App. Pub. No. 20050027070.

In another embodiment, the cells of the invention (e.g., cells made bypracticing the methods of this invention) are readministered (e.g.,injected or engrafted) to a tissue, an organ and/or an individual inneed thereof within, or protected by, a biocompatible, nonimmunogeniccoating, e.g., as on the surface of a synthetic implant, e.g., asdescribed in U.S. Pat. No. 6,969,400, describing e.g., a protocol wherea chimeric protein of this invention can be conjugated to a polyethyleneglycol that has been modified to contain multiple nucleophilic groups,such as primary amino or thiol group.

In one embodiment, the cells of the invention (e.g., cells made bypracticing the methods of this invention; cells comprising codingsequence for chimeric polypeptides of this invention) are readministered(e.g., injected or engrafted) to a tissue, an organ and/or an individualin need thereof using grafting methods as described e.g. by U.S. Pat.Nos. 7,442,390; 5,733,542.

Diagnosing and Treating Anemia

In alternative embodiments, the invention provides methods forameliorating or preventing an anemia, and/or stimulating erythropoiesisand/or erythropoietin (EPO) synthesis, in an individual. In practicingthe invention any diagnostic method for determining when anemia ispresent, or the severity of the anemia, or whether the anemia isresponding to treatment (e.g., responding to administration of achimeric protein of the invention, or administration of a nucleic acidencoding a chimeric protein of the invention), or whether levels of EPOare normal or responding to treatment, can be used.

For example, because alternative embodiments of the invention encompassameliorating or preventing anemia caused by a genetic disorder, aninfection, a dietary disorder or deficiency, a pollutant, a pesticide,herbicide or insecticide, a poison, a venom, a toxin, a biologicalagent, a drug, a cancer or a cancer therapeutic or cancer therapy, thepresence of any of these conditions can be used as a basis fordiagnosing or predicting the possible onset of anemia, and anydiagnostic technique related to any of these conditions, treatments,infections or exposures can be used.

For example, because alternative embodiments of the invention encompassameliorating or preventing anemia caused by a drug-induced anemia;caused by an infection; caused by an iron deficiency; caused by rhesusdisease (hemolytic disease of newborn): caused by sickle-cell disease,thalassemia or Plummer-Vinson syndrome (PVS); a sideroblasticanemia-congenital or acquired; caused by Gaucher's disease; caused by avitamin deficiency; caused by autoimmune hemolytic anemia (AIHA); causedby a cancer; or, caused by heavy metal poisoning or pyridoxinedeficiency, any diagnostic test for any of these conditions, treatments,infections or exposures can be used.

For example, anemia can be diagnosed using complete blood counts, thenumber of red blood cells and/or the hemoglobin level. Automaticcounters that measure the size of the red blood cells, e.g., by flowcytometry, can be used to distinguish between the causes of anemia;noting that in alternative embodiments the compositions and methods ofthe invention are used to ameliorate or prevent microcytic, normocyticor macrocytic forms of anemia. Examination of a stained blood smears,e.g., using a microscope, also can be used. In alternative embodiments,four parameters are used to diagnose or assess anemia: RBC count,hemoglobin concentration, mean corpuscular volume (MCV) and red bloodcell distribution width (RDW); e.g., to access the individual's responseto administration of a chimeric protein of the invention, oradministration of a nucleic acid encoding a chimeric protein of theinvention. Hematocrit, mean corpuscular hemoglobin (or “mean cellhemoglobin”, MCH), and mean corpuscular hemoglobin concentration (orMCHC) also can be calculated, and compared to values adjusted for ageand sex. For example, for adult men a hemoglobin level less than 13.0g/dl (grams per deciliter) is diagnostic of anemia, and for adult women,the diagnostic threshold is below 12.0 g/dl. A normal hematocrit valueis 32 to 36 g/dl.

In alternative embodiments, other tests also can be used, e.g., theerythrocyte sedimentation rate (ESR); the reticulocyte production index(RPI, also called a corrected reticulocyte count); folic acid, serumiron, hepcidin, transferrin, vitamin B12 and/or creatinine levels; e.g.,to access the individual's response to administration of a chimericprotein of the invention, or administration of a nucleic acid encoding achimeric protein of the invention.

In alternative embodiments, methods of this invention can be practicedwith other treatments for anemia, e.g., blood transfusions, hyperbaricmedicine, administration of an EPO, e.g., a recombinant erythropoietin;for example, any form of erythropoietin can be used, including epoetin(e.g., EPOGEN™, PROCRIT™, EPREX™, NEORECORMON™) (Amgen, Thousand Oaks,Calif.), or darbepoetin, a synthetic form of EPO (e.g., ARANESP™)(Amgen, Thousand Oaks, Calif.). In alternative embodiments, methods ofthis invention can be practiced with other treatments for anemia such asadministering a VEGF antagonist, as described e.g., in U.S. Pat. No.7,351,411; administering a hyperglycosylated analog of EPO, as describede.g., in U.S. Pat. No. 7,304,150 or U.S. Pat. No. 7,262,166.

Treating Conditions Response to Increasing EPO Production

In alternative embodiments, the invention also provides methods forincreasing endogenous erythropoietin (EPO) production in an individualin need thereof using compositions of this invention; e.g., includingpharmaceutical or sterile formulations, the chimeric protein of theinvention; liquids, gels, hydrogels, powders or aqueous formulations ofthe invention; vesicles, liposomes, nanoparticles or nanolipid particles(NLP) of the invention; isolated or cultured cells of the invention;nucleic acids of the invention; or vectors, plasmid, recombinant virusor expression vehicle of the invention.

Individuals that can benefit by practicing the compositions and methodsof this invention, e.g., that can benefit (e.g., by treating,ameliorating and/or preventing) from an increase in endogenouserythropoietin (EPO) production, including humans or animals, includee.g., individuals with a peripheral nerve injury; a hemoglobin Hdisease; a testicular torsion-detorsion; post-infarct myocardial damage;or a neurologic disorder; or an increase in endogenous EPO levels wouldact as a neuroprotective, e.g., in low birth weight infants. Inalternative embodiments, the neurologic disorder is ischemic stroke,intracerebral hemorrhage, subarachnoid hemorrhage, traumatic braininjury or Parkinson's disease.

Wounds and inflammations also can be treated or ameliorated bypracticing the compositions and methods of this invention, e.g.,including humans or animals. In alternative embodiments, wounds andinflammations that can benefit from an increase in endogenouserythropoietin (EPO) production by practicing the compositions andmethods of this invention include conjunctivitis; surgical or accidentalwounds; a bedsore; a burn; an inflammation of the skin, mucousmembranes, airways or lungs; an eczema or a skin disorder accompanied bynecrosis, by dermatitis, by psoriasis or by diabetes mellitus. Thepharmaceutical formulation applied can be a topical application, e.g.,in the form of an ointment, a cream, a powder, an emulsion, a gel, aglycerogelatin, a paste, a plaster, a sprayable composition or a lotion.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Compositions and Methods of the Invention areEffective in the Amelioration of Anemia

The data presented herein demonstrates that the compositions and methodsof the invention are effective in the amelioration of anemia.

In one embodiment, each finger of a chimeric protein (DesignedRegulatory Protein, or DRP) of the invention is connected by a flexiblelinker having the amino acid sequence TGEKP, this motif is highlyconserved among C2H2 zinc fingers. Exactly the same scaffold was usedfor each finger except for DNA binding residues. Listed below is areexemplary chimeric polypeptides of this invention: the so-called“EPO1-DRP” (SEQ ID NO:1); and, the so-called “EPO2-DRP” (SEQ ID NO:2):

Amino acid sequence the exemplary DRP-1 of this inventionMGRRRRRRRRRGGGPKKKRKVGGGGSTGEKPYKCPECGKSFS

S

LQ

H      R-CPP      NLS                   Finger-1 QRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHTGEKPYKCPECGKSFS

S

LQ              Finger-2                   Finger-3

HQRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHTGEKPYKCPECGKSFS

S

LQ              Finger-4                   Finger-5

HQRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHGGGGSPRPTDVSLGDELHLDGE              Finger-6                   VP16DVAMAH- ADADLLFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALG             VP16 DYKDDDDK FLAG-tag

Amino acid sequence of the exemplary DRP-2 of this inventionMGRRRRRRRRRGGGPKKKRKVGGGGSTGEKPYKCPECGKSFS

S

LQ

H      R9-CPP     NLS                   Finger-1 QRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHTGEKPYKCPECGKSFS

S

LQ              Finger-2                 Finger-3

HQRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHTGEKPYKCPECGKSFS

S

LQ              Finger-4                 Finger-5

HQRTH- TGEKPYKCPECGKSFS

S

LQ

HQRTHGGGGSPRPTDVSLGDELHLDGE              Finger-6                 VP16DVAMAH- ADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALG             VP16 DYKDDDDK FLAG-tag

Normal human lymphocytes were used, and the cells contacted withchimeric proteins of this invention, the so-called “Designed RegulatoryProteins (DRPs)” of the invention, using a procedure that is the same asdescribed in Tachikawa, K. et al. Proc. Natl. Acad. Sci. USA101:15225-30, 2004. Purified EPO1-DRP (SEQ ID NO:1) or EPO2-DRP (SEQ IDNO:2) protein was incubated with the cells. FIG. 1 illustratesphotomicrograph images that confirms that chimeric proteins (DRPs) ofthis invention can enter mammalian cells, using in this example humanlymphocyte cells: left two panels illustrate a fluorescence image, andright two panels illustrate a phase contrast image; the upper two panelsillustrate detection of green fluorescent protein expressed by a DRP ofthe invention.

FIG. 2 illustrates data from an ELISA assay demonstrating that bothEPO1-DRP (the DRP-1 described above, SEQ ID NO:1) and EPO2-DRP (theDRP-2 described above, SEQ ID NO:2) of this invention when inserted intomammalian cells can enhance secretion of EPO protein in those cells. Forexample, as graphically illustrated in FIG. 2, both EPO1-DRP andEPO2-DRP enhanced secretion of EPO protein in mouse fibroblast cells.FIG. 3 graphically illustrates data of blood EPO in these mice (i.e.,animal administered EPO1-DRP or EPO2-DRP) further demonstrating bothEPO1-DRP and EPO2-DRP of this invention when inserted into mammaliancells can enhance secretion of EPO protein in those cells. Both theexemplary EPO1-DRP and EPO2-DRP caused an enhancement of secretion ofEPO in the mice. In FIG. 3, the “*” indicates exemplary alanine mutants:the figure illustrating amino acid residues necessary for sequencespecific recognition (i.e. −1, +2, +3, and +6 in the each finger domainof the DRP) were replaced with alanine (ala).

Example 2 Designing Compositions of the Invention to Ameliorate Anemia

FIG. 4 illustrates exemplary zinc finger DNA target sequences forchimeric proteins (DRPs) of the invention, including but not limited tothe illustrated DRP-1 and DRP-2 target site illustrated in this figure;“*” indicates the number on the zinc finger target sequence thatrepresents the position relative to the transcription start site. FIG. 5illustrates alternative exemplary zinc finger DNA target sequences forchimeric proteins (DRPs) of the invention, with the correspondingexemplary zinc finger DNA-binding motif; the figure illustratesexemplary zinc finger DNA target subsites 5′ to 3′ and amino acidresidues −1 to +6 (minus one to plus six):

Target subsites Amino acid residues Finger 5′ to 3′ −1 to +6 Zif 1 GAGg(SEQ ID NO: 19) RSSNLQR (SEQ ID NO: 7) Zif 2 TGGc (SEQ ID NO: 20)RSDHLQT (SEQ ID NO: 8) Zif 3 GGGa (SEQ ID NO: 21) RSNHLQR (SEQ ID NO: 9)Zif 4 GGGc (SEQ ID NO: 22) RSDHLQR (SEQ ID NO: 10) Zif 5 T G Tc(SEQ ID NO: 23) TSDHLQT (SEQ ID NO: 11) Zif 6 G TGa (SEQ ID NO: 24)RSNSLQR (SEQ ID NO: 12) Zif 1 GGGt (SEQ ID NO: 25) RSTHLQR(SEQ ID NO: 13) Zif 2 GGGc (SEQ ID NO: 26) RSDHLQR (SEQ ID NO: 14) Zif 3GGGc (SEQ ID NO: 27) RSDHLQR (SEQ ID NO: 15) Zif 4 T TGc (SEQ ID NO: 28)RSDSLQT (SEQ ID NO: 16) Zif 5 G T Ta (SEQ ID NO: 29) TSNSLQR(SEQ ID NO: 17) Zif 6 GGGc (SEQ ID NO: 30) RSDHLQR (SEQ ID NO: 18)

This invention can incorporate any zinc finger-interacting proteinfamily member, or “zif”, including the zif motifs illustrated in FIG. 6,including the exemplary zif-1, zif-2, zif-3, zif-4, zif-5 and/or zif-6.

This invention can incorporate any protein that can specifically bind toa zinc finger target sequence, e.g., as described in U.S. Pat. App. Pub.Nos. 20030082561, 20030134350 and 20040091878 (also WO/2003/062455),describing a context-independent recognition code to design zinc fingerdomains; the code permits identification of an amino acid for positions−1, 2, 3 and 6 of the alpha-helical region of the zinc finger domainfrom four-base pair nucleotide target sequences.

This invention can incorporate any zinc finger binding protein or motif,e.g., as described in U.S. Pat. App. Pub. No. 20080269471; or U.S. Pat.App. Pub. No. 20080182332, describing zinc fingers comprising CCHC zinccoordinating residues; or U.S. Pat. App. Pub. No. 20060246588,describing non-canonical (e.g., non-C₂H₂) zinc finger proteins.

For example, embodiments of the invention include chimeric proteinscomprising one or more or all of the following amino acid zinc fingerDNA binding motifs (see also FIG. 6):

TGEKPYKCPECGKSFSRSSNLQRHQRTH (SEQ ID NO: 31)TGEKPYKCPECGKSFSRSDHLQTHQRTH (SEQ ID NO: 32)TGEKPYKCPECGKSFSRSNHLQRHQRTH (SEQ ID NO: 33)TGEKPYKCPECGKSFSRSDHLQRHQRTH (SEQ ID NO: 34)TGEKPYKCPECGKSFSTSDHLQTHQRTH (SEQ ID NO: 35)TGEKPYKCPECGKSFSRSNSLQRHQRTH (SEQ ID NO: 36)

Chimeric proteins of this invention can be designed to specifically bindto, and modulate (e.g., activate or inhibit) the transcription of, anyEPO gene, for example:

LOCUS   EPO-6\in\p  957 bp  DNA                     6 JUL. 2006COMMENT   CGexDoc “EPO-6 in pET21” ORIGDB|GenBankBASE COUNT         262 a      236 c     267 g    192 t (SEQ ID NO: 37) 1taattttgtt taactttaag aaggagatat accatgggtc gtagacgcag gcgtagacgc 61aggcgtggtg gcggtccgaa gaaaaagcgt aaagtgggcg gtggcggatc cacgggtgag 121aagccgtata aatgtcccga atgtggtaaa agttttagcc gttcgaccca tctgcaacgt 181catcaacgca cccataccgg cgaaaaacca tacaaatgtc cggagtgcgg caaatctttc 241tcgcgcagcg atcatttgca gcgccatcag agaactcaca ctggcgagaa gccctacaag 301tgccccgaat gcgggaagag ctttagtaga tctgatcact tacaaaggca ccagaggacg 361cataccggtg agaagccgta taaatgtccc gaatgtggta aaagttttag cacgtcggac 421agcctgcaaa cccatcaacg cacccatacc ggcgaaaaac catacaaatg tccggagtgc 481ggcaaatctt tctcgcgcag caattcgttg cagcgtcatc agagaactca cactggcgag 541aagccctaca agtgccccga atgcgggaag agctttagta gatctgatca tttacaacgc 601caccagagga cgcatacggg cgagaaaccg tatggtggcg gtggcagccc taggccgacc 661gatgtgagcc tgggcgatga actgcattta gatggcgaag atgtggcgat ggcccatgcg 721gatgccctag acgattttga cctggatatg ttaggcgatg gtgacagccc cggtccgggt 781tttaccccgc atgatagcgc accgtatggt gcgctagata tggcggattt cgaatttgaa 841cagatgttta ccgatgcgct gggtattgat gaatatggcg gtgctagcga ttataaagat 901gacgatgaca aataagcggc cgcactcgag caccaccacc accaccactg agatccgLOCUS   EPO-1\in\p  957 bp  DNA COMMENT (CGexDoc “EPO-1 in pET21” 1 957ORIGDB|GenBank (SEQ ID NO: 38) 1taattttgtt taactttaag aaggagatat accatgggtc gtagacgcag gcgtagacgc 61aggcgtggtg gcggtccgaa gaaaaagcgt aaagtgggcg gtggcggatc cacgggtgag 121aagccgtata aatgtcccga atgtggtaaa agttttagcc gttcgagcaa tctgcaacgt 181catcaacgca cccataccgg cgaaaaacca tacaaatgtc cggagtgcgg caaatctttc 241tcgcgtagcg atcatttgca gacccatcag agaactcaca ctggcgagaa gccctacaag 301tgccccgaat gcgggaagag ctttagtaga tctaatcact tacaacgcca ccagaggacg 361cataccggtg agaagccgta taaatgtccc gaatgtggta aaagttttag ccgctcggac 421catctgcaaa gacatcaacg cacccatacc ggcgaaaaac catacaaatg tccggagtgc 481ggcaaatctt tctcgaccag cgatcacttg cagacgcatc agagaactca cactggcgag 541aagccctaca agtgccccga atgcgggaag agctttagta gatctaatag cttacaacgc 601caccagagga cgcatacggg cgagaaaccg tatggtggcg gtggcagccc taggccgacc 661gatgtgagcc tgggcgatga actgcattta gatggcgaag atgtggcgat ggcccatgcg 721gatgccctag acgattttga cctggatatg ttaggcgatg gtgacagccc cggtccgggt 781tttaccccgc atgatagcgc accgtatggt gcgctagata tggcggattt cgaatttgaa 841cagatgttta ccgatgcgct gggtattgat gaatatggcg gtgctagcga ttataaagat 901gacgatgaca aataagcggc cgcactcgag caccaccacc accaccactg agatccg //

In one embodiment, the chimeric proteins of the invention bind topromoters of human EPO and activate the transcription of EPO; and inalternative embodiments the EPO activated by the chimeric proteins ofthe invention (by transcriptional activation of promoters) is aheterologous EPO gene. In one embodiment a human EPO gene such as (SEQID NO:39) is used:

(SEQ ID NO: 39) 1cccggagccg gaccggggcc accgcgcccg ctctgctccg acaccgcgcc ccctggacag 61ccgccctctc ctccaggccc gtggggctgg ccctgcaccg ccgagcttcc cgggatgagg 121gcccccggtg tggtcacccg gcgcgcccca ggtcgctgag ggaccccggc caggcgcgga 181gatgggggtg cacgaatgtc ctgcctggct gtggcttctc ctgtccctgc tgtcgctccc 241tctgggcctc ccagtcctgg gcgccccacc acgcctcatc tgtgacagcc gagtcctgga 301gaggtacctc ttggaggcca aggaggccga gaatatcacg acgggctgtg ctgaacactg 361cagcttgaat gagaatatca ctgtcccaga caccaaagtt aatttctatg cctggaagag 421gatggaggtc gggcagcagg ccgtagaagt ctggcagggc ctggccctgc tgtcggaagc 481tgtcctgcgg ggccaggccc tgttggtcaa ctcttcccag ccgtgggagc ccctgcagct 541gcatgtggat aaagccgtca gtggccttcg cagcctcacc actctgcttc gggctctggg 601agcccagaag gaagccatct cccctccaga tgcggcctca gctgctccac tccgaacaat 661cactgctgac actttccgca aactcttccg agtctactcc aatttcctcc ggggaaagct 721gaagctgtac acaggggagg cctgcaggac aggggacaga tgaccaggtg tgtccacctg 781ggcatatcca ccacctccct caccaacatt gcttgtgcca caccctcccc cgccactcct 841gaaccccgtc gaggggctct cagctcagcg ccagcctgtc ccatggacac tccagtgcca 901gcaatgacat ctcaggggcc agaggaactg tccagagagc aactctgaga tctaaggatg 961tcacagggcc aacttgaggg cccagagcag gaagcattca gagagcagct ttaaactcag 1021ggacagagcc atgctgggaa gacgcctgag ctcactcggc accctgcaaa atttgatgcc 1081aggacacgct ttggaggcga tttacctgtt ttcgcaccta ccatcaggga caggatgacc 1141tggagaactt aggtggcaag ctgtgacttc tccaggtctc acgggcatgg gcactccctt 1201ggtggcaaga gcccccttga caccggggtg gtgggaacca tgaagacagg atgggggctg 1261gcctctggct ctcatggggt ccaagttttg tgtattcttc aacctcattg acaagaactg 1321aaaccaccaa aaaaaaaaaa

In one embodiment, compositions of the invention (e.g., chimericproteins and/or the nucleic acids that encode them) are administeredwith EPO, e.g., the human EPO as in SEQ ID NO:40:

(SEQ ID NO: 40) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFL RGKLKLYTGEACRTGDR

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A chimeric protein comprising (a) (1) a plurality of (multiple)DNA-binding domains specific for (that can specifically bind to) apromoter and/or another transcriptional regulatory region of anerythropoietin (EPO) gene or a Erythropoietin Stimulating Protein (NESP)gene; (2) at least one nuclear localization peptide (NLP) domain; (3) atleast one cell-penetrating peptide (CPP); and, (4) at least onetranscription activation (TA) domain; or (b) the chimeric protein of(a), comprising an EPO-specific zinc finger DNA-binding domain, or anNESP-specific zinc finger DNA-binding domain.
 2. The chimeric protein ofclaim 1: (a) wherein the chimeric protein, the EPO- or NESP-specificDNA-binding domain, the NLP domain, the CPP domain and/or the TA domaincomprises a recombinant protein, a synthetic protein, a peptidomimetic,a non-natural peptide or a combination thereof; (b) wherein the EPO orNESP gene or transcriptional regulatory region of an EPO or NESP genecomprises: (a) a mammalian EPO or NESP gene or transcriptionalregulatory region or (b) a mouse or a human EPO or NESP gene ortranscriptional regulatory region; (c) wherein the chimeric protein of(b), wherein the transcriptional regulatory region comprises a promoteror an enhancer, an EPO or NESP promoter or EPO or NESP enhancer, or asynthetic promoter; (d) wherein the chimeric protein comprises multiplecopies of the EPO- or NESP-specific DNA-binding domain, the NLP, the CPPand/or the TA domain; (e) wherein the chimeric protein comprises two,three, four, five, six or more EPO- or NESP-specific DNA-binding domainsspecific for (that can specifically bind to) a promoter and/or anothertranscriptional regulatory region of an EPO or NESP gene; (f) whereinthe chimeric protein comprises one, two, three, four, five, six or morenuclear localization peptide (NLP) domains or consensus nuclearlocalization proteins; (g) wherein the chimeric protein comprises one,two, three, four, five, six or more cell-penetrating peptides (CPPs) ;(h) wherein the chimeric protein comprises one, two, three, four, five,six or more TA domains, and/or one or more other functional domains witha histone acetyltransferase (HAT) activity; (i) wherein the chimericprotein of (h), wherein the at least one TA domain comprises a herpessimplex virus (HSV) VP-16 activation peptide domain or a peptide derivedfrom the C-terminal transcription activation domain of β-catenin (FDTDL)(SEQ ID NO:30); (j) wherein the at least one zinc finger DNA-bindingdomain comprises (1) a zinc-finger of the C₂H₂ class; (2) a zinc-fingerof the C₄ class; or (3) a zinc-finger of C₆ class; (k) wherein the atleast one zinc finger DNA-binding domain comprises the consensussequence Cys-X₂₋₄-Cys-X₃-Phe-X₅-Leu-X₂-His-X₃-His (SEQ ID NO:31); (l)wherein the at least one nuclear localization peptide (NLP) domaincomprises (1) an NLP sequence of a large T antigen of the simian virus40 (SV-40), or PKKKRKV (SEQ ID NO:29) (SEQ ID NO:2); (2) a consensussequence fitting B₄ (SEQ ID NO:32), P(B₃X) (SEQ ID NO:33), PXX(B₃X) (SEQID NO:34), B₃(H/P) (SEQ ID NO:35), where B is a basic amino acid, P isproline, H is histidine, X is any amino acid and letters in parenthesescan be in any order; (3) a bipartite NLP comprising two short stretchesof basic amino acids separated by a non-conserved sequence; or (4) acellular nucleoplasm [[̂]] protein KRPAATKKAGQAKKKK (SEQ ID NO:4); (m)wherein the at least one cell-penetrating peptide (CPP) comprises (1) aplurality of polycationic amino acid residues; (2) a plurality ofarginine amino acid residues; or (3) a TAT protein (Trans-actingActivator of Transcription) of a Human Immunodeficiency Virus (HIV-l) ;(n) wherein (1) the at least one TA domain is at least approximately 25%hydrophobic and is linked to the at least one zinc finger DNA-bindingdomain in a manner that does not interfere with the promoter or atranscriptional regulatory binding activity of the zinc finger DNAbinding peptide, and the TA domain is both necessary and sufficient toactivate transcription of the gene; and/or (2) the TA domain is betweenabout 5 to 25 amino acids in length, or is between about 6 to 20 aminoacids in length, or is about 5, 6, 7, 8, 9, 10, 11, 11, 12, 13, 14 or 15amino acids in length; (o) wherein the at least one TA domain comprisesa herpes simplex virus (I-ISV) VP-16 activation peptide domain or apeptide derived from the C-terminal transcription activation domain ofβ-catenin (FDTDL) (SEQ ID NO:30); (p) wherein at least one, or all, ofthe domains and/or chimeric proteins further comprises, or is attachedto, a lipid, a hydrophobic alkane or alkene (olefin) moiety, or apolyethylene glycol (PEG) moiety; (q) wherein at least one, or all, ofthe chimeric proteins further comprises, or is attached to, an epitopepeptide tag or a detectable composition or moiety; (r) wherein thedetectable composition or moiety comprises a phosphoprotein, afluorescent molecule, a fluorescent tagged protein, a radiolabel or aradiolabeled protein; (s) further comprising a small molecule, a hormoneor a cytokine that increases or upregulates erythropoiesis or red bloodcell production in a mammalian; or (t) wherein the chimeric proteincomprises (a) a formulation for subcutaneous, parenteral, topical, oralor local administration, or for aerosol or transdermal administration,or administration by nebulizer; or (b) the chimeric protein of (a),wherein the topical formulation comprises an ointment, a cream, apowder, an emulsion, a gel, a glycerogelatin, a paste, a plaster, asprayable composition or a lotion. 3-21. (canceled)
 22. A compositioncomprising: (a) a plurality of chimeric proteins of claim 1; (b) thecomposition of (a), further comprising a small molecule, a hormone or acytokine that increases or upregulates erythropoiesis or red blood cellproduction in a mammalian; (c) the composition of (a), furthercomprising a synthetic or recombinant erythropoietin; (d) thecomposition of (a), further comprising or formulated as a liquid, gel,hydro gel, powder or aqueous formulation, or a vesicle, liposome,nanoparticle or nanolipid particle (NLP); (e) the composition of (a),further comprising or formulated as or is contained in an isolated orcultured cell, or a mammalian cell, or a human cell, a non-human primatecell, a monkey cell, a mouse cell, a rat cell, a guinea pig cell, arabbit cell, a hamster cell, a goat cell, a bovine cell, an equine cell,an ovine cell, a canine cell or a feline cell; (f) the composition ofany of (a) to (d), further comprising or formulated as or is containedin a pharmaceutical or sterile formulation; or (g) the composition ofany of (a) to (d), further comprising or formulated as or is containedin a product of manufacture. 23-31. (canceled)
 32. A recombinant orsynthetic nucleic acid comprising: (a) a nucleic acid sequence encodingthe chimeric protein of claim 1; (b) the recombinant or syntheticnucleic acid of (a) contained in a vector, plasmid, recombinant virus orexpression vehicle, and optionally the nucleic acid is operativelylinked to a constitutive promoter or an inducible promoter or a promoteronly active in a hematopoietic cell; or (c) the recombinant or syntheticnucleic acid of (a) contained in an isolated or cultured cell, or amammalian cell, or a human cell, a non-human primate cell, a monkeycell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, ahamster cell, a goat cell, a bovine cell, an equine cell, an ovine cell,a canine cell or a feline cell. 33-37. (canceled)
 38. A method forameliorating or preventing an anemia, and/or stimulating erythropoiesisand/or erythropoietin (EPO) synthesis, in an individual comprising: (1)(a) providing: the pharmaceutical or sterile formulation of claim 30;and (b) administering an effective amount of (a) to an individual inneed thereof; (2) the method of (1), wherein the individual in needthereof is a mammalian or a human; (3) the method of (1), wherein theanemia ameliorated or prevented is caused by a genetic disorder, aninfection, a dietary disorder or deficiency, a pollutant, a pesticide,herbicide or insecticide, a poison, a venom, a toxin, a biologicalagent, a drug, a cancer or a cancer therapeutic or cancer therapy; (4)the method of (1), wherein the anemia ameliorated or prevented is amicrocytic, normocytic or macrocytic form of anemia, or the anemiaameliorated or prevented is: a drug-induced anemia; caused by aninfection; caused by an iron deficiency; caused by rhesus disease(hemolytic disease of newborn); caused by sickle-cell disease,thalassemia or Plummer-Vinson syndrome (PVS, also calledPaterson-Brown-Kelly syndrome or sideropenic dysphagia); a sideroblasticanemia-congenital or acquired; caused by Gaucher's disease; caused by avitamin deficiency; caused by autoimmune hemolytic anemia (AIHA); causedby a cancer; or, caused by heavy metal poisoning or pyridoxinedeficiency; (5) the method of (4), wherein the vitamin deficiency is afolate or B 12 deficiency (pernicious anemia o_(f) Addison's anemia);(6) the method of (4), wherein the drug-induced anemia is caused bymethyldopa or fludarabin); (7) the method of (4), wherein the AIHA iscaused by Systemic lupus erythematosus, a drug, Evans syndrome, chroniclymphocytic leukemia or is idiopathic); (8) the method of (4), whereinthe cancer is chronic lymphocytic leukemia, small cell lymphoma (orsmall lymphocytic lymphoma) or a non-Hodgkin's lymphoma; or the anemiais caused by myelophythisis secondary to an acute megakaryoblasticleukemia, a lymphoma, a myeloma or a carcinoma metastatic to bonemarrow); (9) the method of (4), wherein the infection is an EBVinfectious mononucleosis, a Babesiosis infection, or equine infectiousanemia); (10) the method of (4), wherein the cancer therapeutic orcancer therapy is radiotherapy, hormone therapy or chemotherapy); or(11) the method of (4), wherein the heavy metal poisoning is leadpoisoning, mercury poisoning (hydrargaria), copper poisoning, nickelpoisoning, manganese poisoning (manganism) or cadmium poisoning. 39-80.(canceled)